JP2016138117A - Phenothiazine diaminium salts and their use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide phenothiazine diaminium compounds which are useful in the treatment of, for example, Alzheimer's disease.SOLUTION: The present invention provides a compound represented by formula (I) (R, R, R, R, R, R, Rand Rindependently represent Calkyl, halogenated Calkyl or the like).SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates generally to the field of phenothiazine compounds, particularly certain phenothiazine diaminium salts, including their uses and formulations. In some embodiments, the present invention relates to bis (sulfonic acid) salts of diaminophenothiazine compounds such as N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diamine. The compounds of the present invention are useful in the treatment of tauopathy such as Alzheimer's disease (AD).

In order to more fully describe and disclose the present invention and the current state of the art to which the present invention pertains, numerous patents and publications are cited herein. Each of these references is hereby incorporated by reference in its entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. .

  Throughout this specification, including the claims that follow, unless the context requires otherwise, the terms “comprise” and variations such as “comprises” and “comprising” are stated. It is understood that it is meant to encompass any integer or step or group of integers or steps, and is not meant to exclude any other integer or step or group of integers or steps. Let's go.

  It should be noted that the singular forms “a”, “an”, and “the” used herein and in the appended claims include plural referents unless the context clearly dictates otherwise. I must. Thus, for example, reference to “a pharmaceutical carrier” includes a mixture of two or more such carriers, and the like.

  Ranges are often expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, where a value is expressed as an approximation by use of the “about” set forth above, it will be understood that the specific value forms another embodiment.

  Any subtitles herein are included for convenience only and should not be construed as limiting the disclosure in any way.

  Dementia states are often characterized by progressive accumulation of intracellular and / or extracellular deposits of proteinaceous structures such as β-amyloid plaques and neurofibrillary tangles (NFTs) in the brain of affected patients . The appearance of these lesions is highly correlated with pathological neurofibrillary degeneration and brain atrophy, and cognitive impairment (eg, Mukaetova-Ladinska, EB etal., 2000, Am. J. Pathol., Vol 157, No. 2, see pp. 623-636).

In Alzheimer's disease, senile plaques and NFTs contain paired helical fibrils (PHFs), the main component of which is the microtubule-binding protein, tau (eg, Wischik et al., 1988, PNAS USA, Vol. 85, pp. 4506-4510). The plaques also contain extracellular β-amyloid fibrils derived from abnormal processing of amyloid precursor protein (APP) (see, eg, Kang et al., 1987, Nature, Vol. 325, p. 733). . ( 'Neurobiology of Alzheimer'sDisease', 2 nd Edition, 2000, Eds. Dawbarn, D and Allen, SJ, TheMolecular and Cellular Neurobiology Series, Bios Scientific Publishers, in Oxford) Wischik et al paper by the neurodegenerative dementia We are investigating the possible role of tau protein in pathogenesis. Loss of the normal form of tau, accumulation of pathological PHFs, and loss of synapses in the middle frontal cortex are all correlated with associated cognitive impairment. Furthermore, both synaptic and pyramidal loss correlated with morphological measures of tau-responsive neurofibrillary lesions, which in Alzheimer's disease are polymerized from soluble forms of the tau protein pool at the molecular level Comparable to almost total redistribution into form (ie PHFs).

  Tau exists as an alternative splicing isoform and contains 3 or 4 copies of a repetitive sequence corresponding to the microtubule binding domain (eg Goedert, M., et al., 1989, EMBOJ., Vol. 8, pp. 393 -399; Goedert M., et al., 1989, Neuron, Vol. 3, pp. 519-526). Tau in PHFs is proteolytically processed to the core domain (eg Wischik, CM, et al., 1988, PNAS USA, Vol. 85, pp. 4884-4888; Wischik et al., 1988, PNAS USA , Vol. 85, pp. 4506-4510; see Novak, M., et al., 1993, EMBO J., Vol. 12, pp. 365-370), which is a phase-shifted version of the repetitive domain Only three iterations are involved in stable tau-tau interactions (see, for example, Jakes, R., et al., 1991, EMBOJ., Vol. 10, pp. 2725-2729) . Once formed, PHF-like tau aggregates act as seeds for further capture and provide a template for proteolytic processing of full-length tau protein (see, eg, Wischik et al. , 1996, PNAS USA, Vol. 93, pp. 11213-11218).

  The phase shift observed in repetitive domains incorporated into PHFs suggests that the repetitive domains undergo induced conformational changes during uptake into fibrils. It is expected that during the onset of AD, this conformational change can be initiated by the binding of tau to pathological substrates such as damaged or mutated membrane proteins (eg, Wischik, CM et al. 1997). , in "Microtubule-assosiated proteins: modifications in disease", Eds. Avila, J., Brandt, R. and Kosik, KS (see Harwood Academic Publishers, Amsterdam) pp. 185-241).

PHFs first assemble in the process of their formation and accumulation, possibly forming amorphous aggregates in the cytoplasm from early tau oligomers that are cleaved before or during PHF assembly (eg Mena , R., et al., 1995, ActaNeuropathol., Vol. 89, pp. 50-56; Mena, R., et al., 1996, Acta Neuropathol., Vol. 91, pp. 633-641 about). These fibrils then subsequently form classic intracellular NFTs. In this state, PHFs consist of a fluffy outer covering comprising a cut-out tau core and full-length tau (see, eg, Wischik et al., 1996, PNAS USA, Vol. 93, pp. 11213-11218). ). This assembly process is exponential and consumes an intracellular pool of normal functional tau and induces the synthesis of new tau to compensate for the deficiency (eg Lai, RYK et al., 1995, Neurobiology of Ageing, Vol. 16, No. 3, pp. 433-445). Eventually, neuronal dysfunction progresses towards cell death, leaving behind an extracellular NFT. Cell death is highly correlated with the number of extracellular NFTs (eg, Wischik et al., 'Neurobiology ofAlzheimer's Disease', 2 nd Edition, 2000, Eds. Dawbarn, D. and Allen, SJ, The Molecular and Cellular (See Neurobiology Series, Bios Scientific Publishers, Oxford). When tangles are pushed into the extracellular space, the fuzzy outer covering of neurons is rapidly lost, and the corresponding loss of N-terminal tau immunoreactivity is associated with tau immunoreactivity associated with the PHF core. Are stored (see, for example, Bondareff, W. et al., 1994, J. Neuropath.Exper. Neurol., Vol. 53, No. 2, pp. 158-164).

Diaminophenothiazine compound (methylene blue (MB); methylthionine chloride; tetramethylthionine chloride; 3,7-bis (dimethylamino) phenothiazine-5-ium chloride; Cl. Basic Blue 9; tetramethylthionine chloride; 3, 7-bis (dimethylamino) phenazathionium chloride; Swiss blue; CI 52015; CI Solvent Blue 8; aniline violet; and also known as Urorene Blue®) methylthioninium chloride ( MTC) is a low molecular weight (319.86), water soluble, tricyclic organic compound of the formula:

  Methylthioninium chloride (MTC) is a well-known phenothiazine dye and redox indicator, as an optical probe in biophysics, as an intercalator in nanoporous materials, as a redox mediator, and also in photoelectrochromic imaging Have been used.

  Methylthioninium chloride (MTC) and other diaminophenothiazines have been described as inhibitors of protein aggregation in diseases where the protein pathologically aggregates.

  In particular, diaminophenothiazines containing MTC have been shown to inhibit tau protein aggregation, disrupt the structure of PHFs, and reverse the proteolytic stability of the PHF core (eg, PCT International Patent Application Publication No. WO 96 / 30766, Hofmann-La Roche). Such compounds have been disclosed for use in the treatment or prevention of various diseases including Alzheimer's disease.

  PCT International Patent Application Publication No. WO 2007/110630 (Wis Ta Laboratories Ltd) is also related to MTC, including ETC, DEMTC, DMETC, DEETC, MTZ, ETZ, MTI, MTILHI, ETI, ETLHI, MTN, and ETN Certain diaminophenothiazine compounds are disclosed and are useful, for example, as agents in the treatment of Alzheimer's disease.

  In addition, PCT International Patent Application Publication No. WO 2005/030676 (The University of the University of Aberdeen) examines their use in the diagnosis and treatment of radiolabeled phenothiazine and tauopathy.

  Methylthioninium chloride (MTC) has also been disclosed for other medical applications. For example, it is currently used to treat methemoglobinemia, a condition that occurs when blood cannot carry oxygen to the place where it is needed in the body. MTC can also be used as a medical dye (eg, to color certain parts of the body before or during surgery); (eg, as an indicator dye to detect certain compounds present in the urine). It is also used as a diagnostic agent; as a mild urinary disinfectant; as a mucosal surface irritant; as a therapeutic and prophylactic agent for kidney stones; and in the diagnosis and treatment of melanoma.

  MTC is used alone to treat malaria (see, eg, Guttmann, P. and Ehrlich, P., 1891, “Uber die wirkung des methylenblau bei malaria,” Berl. Klin. Woschenr., Vol. 28, pp. 953-956) or in combination with chloroquine (eg Schirmer, H., et al., 2003, "Methylene blue as an antimalarial agent," Redox Report, Vol. 8, pp.272-275; Rengelshausen, J., et al., 2004, “Pharmacokinetic interaction of chloroquine and methylene blue combination against malaria,” European Journal of Clinical Pharmacology, Vol. 60, pp. 709-715).

  MTC (named Virostat®, from Bioenvision Inc., New York) has also demonstrated potent in vitro virucidal activity. Specifically, Virostat® is effective against viruses such as HIV and West Nile virus in clinical tests. Virostat (R) is also currently in clinical trials for the treatment of chronic hepatitis C, a viral infection of the liver. This virus, HCV, is a major cause of acute hepatitis and chronic liver disease including cirrhosis and liver cancer.

  MTC can also prevent replication of nucleic acids (DNA or RNA) when used in combination with light. Plasma, platelets and red blood cells do not contain nuclear DNA or RNA. When MTC is introduced into the blood component, it moves into the nucleic acid structure after traversing the bacterial cell wall or viral membrane. When activated by light, the compounds bind to viral or bacterial pathogen nucleic acids and prevent DNA or RNA replication. Because MTC can inactivate pathogens, it has the potential to reduce the risk of pathogen transmission that would remain undetected by testing.

  In the United States, oral and parenteral formulations of MTC have been marketed, usually under the name Uroline Blue®.

The reduced (“leuco”) form MTC, phenothiazine-5-ium salt, is considered to be the “reduced form”, the corresponding 10H-phenothiazine compound, N, N, N ′, N′-tetramethyl-10H— It is considered “oxidized” to phenothiazine-3,7-diamine.

  It is known that the “reduced form (or“ leuco ”form)” is unstable and can be easily and rapidly oxidized to give the corresponding “oxidized” form.

  May et al. (Am J Physiol Cell Physiol, 2004, Vol. 286, pp. C1390-C1398) shows that human erythrocytes sequentially reduce and take up MTC; that MTC itself is not taken up by the cell; Is the reduced form of MTC; the uptake rate is enzyme dependent; and both MTC and reduced MTC are concentrated in the cell (reduced MTC is Equilibrated to form MTC).

  MTC and similar drugs are absorbed in the gastrointestinal tract and enter the bloodstream. Unabsorbed drug penetrates down the gastrointestinal tract to the distal gut. One important undesirable side effect is the action of unabsorbed drugs in the distal gut, such as sensitization of the distal gut and / or antimicrobials of unabsorbed drugs against the flora in the distal gut, both leading to diarrhea. Action. Therefore, it is desirable to minimize the amount of drug that penetrates down to the distal gut. By increasing the uptake of the drug in the gastrointestinal tract (ie, by increasing the bioavailability of the drug), the dosage can be reduced and undesirable side effects such as diarrhea can be improved.

  Because it is the reduced form of MTC that is taken up by the cells, it may be desirable to administer the reduced form to the patient. This may also reduce reliance on the rate-limiting step of enzymatic reduction.

  PCT International Patent Application Publication No. WO 02/055720 (The University of the University of Aberdeen) discloses the use of certain reduced forms of diaminophenothiazine for the treatment of protein-aggregating diseases, primarily tauopathy. .

  PCT International Patent Application Publication No. WO 2007/110627 (Wis Ta Laboratories Ltd) is a class of 3,7-diamino-10H-phenothiazinium that is effective as a drug or prodrug for the treatment of diseases including Alzheimer's disease. Disclose salts. With respect to MTC, these compounds are also in the “reduced” or “leuco” form. These also included the following salts:

Despite providing certain benefits compared to using MTC, LMT. The synthesis of 2HCl may result in CH ₃ Cl trapped within the crystal. Since CH ₃ Cl is toxic and its level needs to be kept below a safe level, it needs to be removed later.

  Furthermore, LMT. 2HBr contains bromine ions. Bromine is less desirable in principle because it is toxic at either high levels or chronic medication, and at low levels it causes side effects such as confusion.

  It can therefore be considered that it would contribute to the technology to provide further salts of methylthioninium compounds having one or more desirable properties compared to those known.

  Furthermore, providing new formulations of methylthioninium compounds that improve the stability, absorption, and / or their effectiveness as therapeutic agents would contribute to the technology.

SUMMARY OF THE INVENTION Here we have identified a new class of stable phenothiazinediaminium compounds that have improved properties compared to the previously disclosed diaminophenothiazine compounds and salts.

  The properties of the compounds are described below, whereby in preferred embodiments, the present invention provides one or more improved physical, pharmacokinetic, biochemical or other beneficial properties. Can be provided.

  In other embodiments, we also provide new formulations of 3,7-diamino-10H-phenothiazinium salt.

  In one aspect, the present invention provides certain compounds, specifically certain phenothiazinediaminium compounds described herein.

  The compound has the following general formula (I):

{Where
R ¹ and R ⁹ are each independently selected from —H, C _1-4 alkyl, C _2-4 alkenyl and halogenated C _1-4 alkyl;
R ^3NA and R ^3NB are each independently selected from —H, C _1-4 alkyl, C _2-4 alkenyl and halogenated C _1-4 alkyl;
R ^7NA and R ^7NB are each independently selected from —H, C _1-4 alkyl, C _2-4 alkenyl and halogenated C _1-4 alkyl;
Furthermore, where
R ^A and R ^B are each independently selected from C _1-4 alkyl, halogenated C _1-4 alkyl, and C _6-10 aryl; or R ^A and R ^B are linked to R ^AB Forming a group, wherein R ^AB is selected from C _1-6 alkylene and C _6-10 arylene}
And a pharmaceutically acceptable salt thereof.

  Another aspect of the invention relates to a method for synthesizing the above compounds.

  Another aspect of the present invention pertains to pharmaceutical compositions comprising a compound described herein and a pharmaceutically acceptable carrier or diluent.

  Another aspect of the invention pertains to methods for preparing pharmaceutical compositions comprising mixing a compound described herein and a pharmaceutically acceptable carrier or diluent.

  Another aspect of the present invention is a solid agent comprising a compound described herein and further comprising at least one diluent suitable for dry compression and optionally one or more other excipients. In the form of a pharmaceutical composition.

  Another aspect of the present invention is a process for the manufacture of a pharmaceutical composition by a dry compression process, wherein the composition comprises a compound as described herein, at least one diluent suitable for dry compression, and optionally The above method is a solid dosage form comprising one or more other excipients.

  Another aspect of the invention is a free flowing cohesive comprising a compound described herein, at least one diluent suitable for dry compression, and optionally one or more other excipients. The above powder, wherein the powder is compressible into a solid dosage form.

  Another aspect of the invention is a method of reversing and / or inhibiting protein aggregation (eg, tau protein, synuclein, etc.), eg, protein aggregation associated with neurodegenerative disease and / or clinical dementia Wherein said method comprises contacting said protein with an effective amount of a compound or composition described herein. Such methods can be performed in vitro or in vivo.

  Another aspect of the present invention is a method of treating or preventing a disease state in a subject, wherein said subject is treated with a prophylactically or therapeutically effective amount of a compound described herein, preferably a pharmaceutical composition, preferably In the form of a solid dosage form pharmaceutical composition as further described herein, relates to the above method comprising administering.

  Another aspect of the present invention pertains to a compound or composition described herein for use in a method of treatment or prevention of the human or animal body (such as a disease state) by therapy.

  Another aspect of the present invention pertains to the use of a compound or composition described herein in the manufacture of a medicament for use in the treatment or prevention of a disease state.

In some embodiments, the disease state is a protein aggregating disease.
In some embodiments, the disease state is a tauopathy, eg, a neurodegenerative tauopathy, eg, Alzheimer's disease or other disease described below.
In some embodiments, the disease state is skin cancer, eg, melanoma.
In some embodiments, the disease state is a viral, bacterial or protozoal disease state such as hepatitis C, HIV, West Nile virus (WNV), or malaria.

  Another aspect of the invention is a method for inactivating pathogens in a sample (such as a blood or plasma sample) comprising introducing a compound or composition described herein into the sample and It relates to the above method comprising the step of exposing the sample to light.

  Another aspect of the present invention is the following: (a) a compound described herein, preferably provided as a pharmaceutical composition in a suitable container and / or with a suitable packaging; and (b) It relates to a kit comprising instructions for use, eg written instructions on how to administer the compound or composition.

  As will be appreciated by those skilled in the art, the features and preferred embodiments of one aspect of the invention also relate to other aspects of the invention.

FIG. 1 shows a ¹ H NMR spectrum of a representative compound of the present invention (LMT.2MsOH) at 600 MHz in deuterated methanol (CD ₃ OD). FIG. 2 shows LMT.1 at a frequency of 100.56 MHz in CD ₃ OD. 1 shows a ¹³ C NMR spectrum of 2MsOH. FIG. 3 shows LMT.1 in a CD ₃ OD at a frequency of 100.56 MHz. 2 shows the DEPT-135 spectrum of 2MsOH. FIG. 4 shows the LMT.1 frequency at a frequency of 100.56 MHz in the CD ₃ OD. 2 shows the HSQC spectrum of 2MsOH. FIG. 5 shows the LMT.D at a frequency of 100.56 MHz in CD ₃ OD. The expanded part of the HSQC spectrum of 2MsOH is shown. FIG. 6 shows LMT. 2 shows the infrared (FT-IR) spectrum of 2MsOH (KBr). FIG. 7 shows LMT. 2 shows an electron impact (EI) mass spectrum of 2MsOH. FIG. 8 shows LMT. 2 shows an electrospray ionization (ESI) mass spectrum of 2MsOH. FIG. 9 shows LMT. 2 shows the UV / Vis spectrum of 2MsOH. FIG. 10 shows LMT. The HPLC trace for 2MsOH is shown. FIG. 11 is a graph showing LMT. 2 shows a powder X-ray diffractogram for 2MsOH. FIG. 12 shows crystalline LMT. 2 shows the FT-Raman spectrum for 2MsOH. Strongest ^{signal, 1615cm -1, 1588cm -1,} was found in 1258cm ^-1, and 1042cm ^-1. FIG. 13 shows crystalline LMT. 2 shows the thermogravimetric profile for 2MsOH. Constant weight until the start of decomposition at 240 to 270 ° C. was detected by TG and TG-FTIR. FIG. 14 shows crystalline LMT. Figure 2 shows differential scanning calorimetry for 2MsOH. Degradation occurred immediately following a sharp melting point at 271 ° C. (ΔH = 87 J / g). FIG. 15a shows crystalline LMT., Measured at 25 ° C. with a scan rate of 5% / hour. 2 shows a dynamic water vapor adsorption (DVS) curve for 2MsOH. The horizontal dashed line indicates the step of taking up one equivalent of water. A stable weight of the sample (less than 0.5% weight change) was observed in the relative humidity (rh) range between 0% and 70%. This r. h. At higher, water uptake increased rapidly and finally the sample was deliquescent. After drying, the water content is 50% r. h. Again reduced to about 4 equivalents. For comparison, the DVS curve of crystalline dihydrochloride (LMT.2HCl) is shown as a dashed line and the DVS curve of dihydrobromide (LMT.2HBr) is shown as a dotted line. FIG. 15b shows crystalline LMT., Measured at 25 ° C. with a scan rate of 5% / hour. 2 shows a dynamic water vapor adsorption (DVS) curve for 2MsOH. The horizontal dashed line indicates the step of taking up one equivalent of water. A stable weight of the sample (less than 0.5% weight change) was observed in the relative humidity (rh) range between 0% and 70%. This r. h. At higher, water uptake increased rapidly and finally the sample was deliquescent. After drying, the water content is 50% r. h. Again reduced to about 4 equivalents. For comparison, the DVS curve of crystalline dihydrochloride (LMT.2HCl) is shown as a dashed line and the DVS curve of dihydrobromide (LMT.2HBr) is shown as a dotted line. FIG. 15c shows LMT. Figure 8 shows a dynamic water vapor adsorption (DVS) curve for 2MsOH as a function of time. Relative humidity is also shown (right axis). The horizontal dashed line indicates the step of taking up one equivalent of water. FIG. 16 shows LMT. 2MsOH (left) and recrystallized LMT. A polarization micrograph for 2MsOH (right) is shown. Crystals of size less than 100 μm were obtained by recrystallization from 2-PrOH / water. Crystals are irregularly shaped. FIG. 17a shows the X-ray crystal structure of LMTEsOH. FIG. 17b shows LMT. The X-ray crystal structure of EDSA is shown. FIG. 17c shows LMT. 2 shows the X-ray crystal structure of 2MsOH. FIG. 18 shows LMT. 2HBr, LMT. 2HCl and LMT. 2 shows a comparison of MT concentration plasma concentrations over time in pigs after administration of 2MsOH. FIG. 19 is a schematic of the equipment used in the dissolution test (see Formulation Example 12).

Detailed Description of the Invention The inventors have found that phenothiazinediaminium compounds have desirable physical properties or other properties and / or surprisingly improved activity compared to the previously disclosed diaminophenothiazine compounds and salts. A new class was identified.

  In other embodiments, they further provided novel formulations of phenothiazine dianiumium compounds, including but not limited to the above classes.

In general terms, unless otherwise required by context, the compounds of the invention can be described as bis (sulfonate) salts (or bis (sulfonic acid) salts) of 3,7-diamino-10H-phenothiazine compounds. . In other words, the compound is a salt of the corresponding 3,7-diamino-10H-phenothiazine compound and an organic sulfonic acid.

  More specifically, the compounds of the present invention have the general formula:

{Where
R ¹ , R ⁹ , R ^3NA , R ^3NB , R ^7NA and R ^7NB are as defined above}
The bis (sulfonate) salt of the compound of

  In some embodiments, the salt is a bis (alkyl sulfonate) salt or a bis (aryl sulfonate) salt.

  In some embodiments, the salt is a bis (methanesulfonate) salt, bis (ethanesulfonate) salt, bis (p-toluenesulfonate) salt, bis (benzenesulfonate) salt, ethanedisulfonate salt, propanedisulfonate salt. Or a naphthalene disulfonate salt.

In some embodiments, the salt is a bis (methanesulfonate) salt (which may also be referred to as a bis (mesylate) salt).
In some embodiments, the salt is a bis (ethane sulfonate) salt (which may also be referred to as a bis (esylate) salt).
In some embodiments, the salt is a bis (p-toluenesulfonate) salt (which may also be referred to as a bis (tosylate) salt).
In some embodiments, the salt is a bis (benzenesulfonate) salt.
In some embodiments, the salt is an ethane disulfonate salt.
In some embodiments, the salt is a propane disulfonate salt.
In some embodiments, the salt is a naphthalene disulfonate salt, preferably a naphthalene-1,5-disulfonate salt.

In other words, the compounds of the present invention comprise a 3,7-diamino-10H-phenothiazine compound, such as those shown above, and two organic sulfonic acid moieties (R ^A SO ₃ H and R ^B SO ₃ H). It can be considered as a product obtained from the reaction. The two organic sulfonic acid moiety, optionally, the same (i.e., R ^A and R ^B are linked) may be present on the molecule.

  In some embodiments, the compounds of the present invention have the following general formula (I):

{Where
R ¹ and R ⁹ are each independently selected from the following: —H, C _1-4 alkyl, C _2-4 alkenyl, and halogenated C _1-4 alkyl;
R ^3NA and R ^3NB are each independently selected from the following: —H, C _1-4 alkyl, C _2-4 alkenyl, and halogenated C _1-4 alkyl;
R ^7NA and R ^7NB are each independently selected from the following: —H, C _1-4 alkyl, C _2-4 alkenyl, and halogenated C _1-4 alkyl;
Furthermore, here
R ^A and R ^B are each independently selected from the following: C _1-4 alkyl, halogenated C _1-4 alkyl, and C _6-10 aryl; or R ^A and R ^B are linked To form a R ^AB group, where R ^AB is selected from: C _1-6 alkylene and C _6-10 arylene}
And the pharmaceutically acceptable salts, solvates and hydrates thereof.

  In the present specification, the compound of the present invention is represented by a general formula showing the structure of a 3,7-diamino-10H-phenothiazine compound which is a protonated form of a 3,7-diamino group.

The resulting doubly positively charged species binds to two sulfonate counterion moieties (which may optionally be present on the same molecule, ie, R ^A and R ^B are linked):

  However, as will be appreciated by those skilled in the art, the same salt can be represented, for example, in other ways as well:

Further Definitions and Preferences The term “C _1-4 alkyl” as used herein is a hydrocarbon having 1 to 4 carbon atoms, which may be aliphatic or alicyclic, or a combination thereof. It relates to a monovalent moiety obtained by removing a hydrogen atom from a compound.

Similarly, the term “C _2-4 alkenyl” refers to the removal of a hydrogen atom from a C _2-4 alkene compound (ie, a hydrocarbon compound containing at least one double bond and 2 to 4 carbon atoms). Relates to the monovalent part obtained by.

The term “C _1-6 alkylene” as used herein refers to two hydrogen atoms of an aliphatic linear hydrocarbon compound having from 1 to 6 carbon atoms, both from the same carbon atom, or Relates to a bidentate moiety obtained by removal from each of two different carbon atoms.

In some embodiments, the C _1-4 alkyl group is a straight chain C _1-4 alkyl group such as: -Me, -Et, -nPr, -iPr, -nPr and n-Bu; -iPr, It can be selected from branched C _3-4 alkyl groups such as -iBu, -sBu, and -tBu; and cyclic C _3-4 alkyl groups such as -cPr and -cBu.

In some _{embodiments, C 2-4} alkenyl group, be chosen from linear _{C 1-4} alkenyl group such as -CH = CH ₂ (vinyl) and _{-CH 2 -CH} = CH 2 (allyl) it can.

In some embodiments, the halogenated C _1-4 alkyl group can be selected from the following: —CF ₃ , —CH ₂ CF ₃ , and —CF ₂ CF ₃ .

The term “C _6-10 aryl” as used herein relates to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring of a C _6-10 aromatic compound, wherein the compound is It has a ring, or 2 (or more fused) rings or more and has from 6 to 10 ring atoms, wherein at least one of the above rings is an aromatic ring.

The term “C _6-10 arylene” as used herein relates to a bidentate moiety obtained by removing two hydrogen atoms from an aromatic compound having from 6 to 10 carbon atoms.

In some embodiments, the C _6-10 aryl group can be selected from C _6-10 carboaryl groups such as phenyl and naphthyl.

In some embodiments, the C _6-10 arylene group can be selected from phenylene and naphthylene.

The C _1-4 alkyl and C _1-6 alkylene groups can be unsubstituted or optionally, for example, halo (eg, F, Cl, Br or I), amino (eg, —NH ₂ , —NHR, or —NR ₂ , wherein each R is independently C _1-4 alkyl), hydroxy (—OH), alkoxy (—OR, where R is independently C _1-4 alkyl) And may be substituted with one or more groups selected from nitro (—NO ₂ ) and the like.

The C _6-10 aryl and C _6-10 arylene groups may be unsubstituted or optionally halogenated C _1-4 such as, for example, C _1-4 alkyl such as —Me, —CF _{3 and the} like. Alkyl, halo (eg, F, Cl, Br, or I), amino (eg, —NH ₂ , —NHR, or —NR ₂ , wherein each R is independently C _1-4 alkyl), It may be substituted with one or more groups selected from hydroxy (—OH), alkoxy (—OR, wherein R is independently C _1-4 alkyl), nitro (—NO ₂ ), and the like.

R ^A and R ^B groups R ^A and R ^B are each independently selected from the following: C _1-4 alkyl, halogenated C _1-4 alkyl, and C _6-10 aryl, or R ^A and R ^B is linked to form a R ^AB group, where R ^AB is selected from the following: C _1-6 alkylene and C _6-10 arylene.

In some embodiments, R ^A and R ^B are each independently selected from the following: C _1-4 alkyl, halogenated C _1-4 alkyl, and C _6-10 aryl.

In some embodiments, R ^A and R ^B are each independently C _1-4 alkyl.
In some embodiments, R ^A and R ^B are each independently selected from Me, Et, nPr, iPr, nBu, iBu, tBu.
In some embodiments, R ^A and R ^B are each independently selected from Me and Et.

In some embodiments, R ^A and R ^B are each independently C _6-10 aryl.
In some embodiments, R ^A and R ^B are each independently selected from benzene, 1-naphthalene, 2-naphthalene and p-toluene.

In some embodiments, R ^A and R ^B are each independently selected from Me, Et, benzene, and p-toluene.

In some embodiments, R ^A and R ^B are the same.
In some embodiments, R ^A and R ^B are different.

In some embodiments, R ^A and R ^B are the same and are independently Me. In that case, the compound is called diaminophenothiazine bis (methanesulfonate) salt and is represented by the general formula (Ia):

In some embodiments, R ^A and R ^B are joined to form a R ^AB group.

  In these embodiments, the compounds of the invention are instead represented by the following general formula Ib:

{Where
R ^AB is selected from C _1-6 alkylene and C _6-10 arylene}
Can be represented by:

In some embodiments, R ^AB is a C _1-6 alkylene group.
In some ^{embodiments, R AB} is the _{following: -CH 2 -, - CH 2} CH 2 -, - CH 2 CH 2 CH 2 -, - CH 2 CH 2 CH 2 CH 2 -, - CH 2 CH _A C _1-6 alkylene group selected from ₂ CH ₂ CH ₂ CH ₂ — and —CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ —.
In some embodiments, R ^AB is a C _1-6 alkylene group selected from methylene (—CH ₂ —), ethylene (—CH ₂ CH ₂ —) and propylene (—CH ₂ CH ₂ CH ₂ —). It is.
In some embodiments, R ^AB is ethylene.

In some embodiments, R ^AB is a C _6-10 arylene group.

In some embodiments, R ^AB is a C _6-10 arylene group selected from phenylene and naphthylene.

In some embodiments, R ^AB is phenylene.
In some embodiments, R ^AB is selected from 1,2-phenylene, 1,3-phenylene, and 1,4-phenylene.
In some embodiments, R ^AB is phenylene optionally substituted with one or more substituents selected from C _1-4 alkyl, halogenated C _1-4 alkyl, halo, and the like.

In some embodiments, R ^AB is naphthylene.
In some embodiments, R ^AB is 1,2-naphthylene, 1,3-naphthylene, 1,4-naphthylene, 1,5-naphthylene, 1,6-naphthylene, 1,7-naphthylene and 1,8. -Selected from naphthylene.
In some embodiments, R ^AB is: 1,5-naphthylene, ie

  And 1,8-naphthylene, ie

  Chosen from.

In some embodiments, R ^AB is naphthylene optionally substituted with one or more substituents selected from C _1-4 alkyl, halogenated C _1-4 alkyl, halo, and the like.

R ¹ and R ⁹ groups In some embodiments, R ¹ and R ⁹ are each independently —H, —Me, —Et, or —CF ₃ .
In some embodiments, R ¹ and R ⁹ are each independently —H, —Me, or —Et.

In some embodiments, R ¹ and R ⁹ are the same.
In some embodiments, R ¹ and R ⁹ are different.

In some embodiments, R ¹ and R ⁹ are each independently —H.
In some embodiments, R ¹ and R ⁹ are each independently -Me.
In some embodiments, R ¹ and R ⁹ are each independently -Et.

R ^3NA and R ^3NB groups R ^3NA and R ^3NB are each independently selected from the following: —H, C _1-4 alkyl, C _2-4 alkenyl, and halogenated C _1-4 alkyl.

In some embodiments, R ^3NA and R ^3NB are each independently selected from the following: C _1-4 alkyl, C _2-4 alkenyl, and halogenated C _1-4 alkyl.

In some ^{embodiments, R 3NA} and ^{R 3NB} are each independently, -Me, -Et, -nPr, -nBu , -CH 2 -CH = CH 2, or -CF _3.

In some embodiments, R ^3NA and R ^3NB are each independently —Me, —nPr, —nBu, —CH ₂ —CH═CH ₂ , or —CF ₃ .

In some embodiments, R ^3NA and R ^3NB are each independently -Me, or -Et.

In some embodiments, R ^3NA and R ^3NB are the same.
In some embodiments, R ^3NA and R ^3NB are different.

In some embodiments, R ^3NA and R ^3NB are each independently -Me.

R ^7NA and R ^7NB groups R ^7NA and R ^7NB are each independently selected from the following: —H, C _1-4 alkyl, C _2-4 alkenyl, and halogenated C _1-4 alkyl.

In some embodiments, R ^7NA and R ^7NB are each independently selected from the following: C _1-4 alkyl, C _2-4 alkenyl, and halogenated C _1-4 alkyl.

In some ^{embodiments, R 7NA} and ^{R 7NB} are each independently, -Me, -Et, -nPr, -nBu , -CH 2 CH = CH 2, or -CF _3.

In some embodiments, R ^7NA and R ^7NB are each independently —Me, —nPr, —nBu, —CH ₂ CH═CH ₂ , or —CF ₃ .

In some embodiments, R ^7NA and R ^7NB are each independently -Me or -Et.

In some embodiments, R ^7NA and R ^7NB are the same.
In some embodiments, R ^7NA and R ^7NB are different.

In some embodiments, R ^7NA and R ^7NB are each independently -Me.

R ^3NA , R ^3NB , R ^7NA and R ^7NB groups In some embodiments:
R ^3NA and R ^3NB are each independently C _1-4 alkyl, C _2-4 alkenyl, or halogenated C _1-4 alkyl;
R ^7NA and R ^7NB are each independently C _1-4 alkyl, C _2-4 alkenyl, or halogenated C _1-4 alkyl.

In some embodiments:
R ^3NA and ^{R 3NB} independently, -Me, -Et, -nPr, _-nBu, be -CH ₂ -CH = CH 2 or -CF _3,;
R ^7NA and R ^7NB are each independently —Me, —Et, —nPr, —nBu, —CH ₂ —CH═CH ₂ , or —CF ₃ .

In some embodiments,
R ^3NA and R ^3NB are each independently -Me or -Et;
R ^7NA and R ^7NB are each independently -Me or -Et.

In some embodiments, R ^3NA and R ^3NB and R ^7NA and R ^7NB are all the same.
In some embodiments, R ^3NA and R ^3NB and R ^7NA and R ^7NB are the same and are all -Me or all -Et.

In some embodiments, R ^3NA and R ^3NB and R ^7NA and R ^7NB are the same and are all -Me.

Salts and Solvates The compounds described herein are themselves salts, but they can also be provided in the form of a mixed salt (ie, a compound of the invention in combination with another salt). Such mixed salts are intended to be encompassed by the term “and pharmaceutically acceptable salts thereof”. Unless stated otherwise, a reference to a particular compound also includes its salts.

  The compounds of the invention can also be provided in the form of solvates or hydrates. As used herein, the term “solvate” means, in the conventional sense, a complex of a solute (such as a compound, salt of a compound, etc.) and a solvent. When the solvent is water, the solvates are conveniently referred to as hydrates such as monohydrate, dihydrate, trihydrate and the like. Unless otherwise specified, a reference to a compound also includes its solvate or hydrate form.

  Of course, solvates or hydrates of the salts of the compounds are also encompassed by the present invention.

Isotopic diversity In some embodiments, one or more carbon atoms of the compound is ¹¹ C, ¹³ C, or ¹⁴ C.
In some embodiments, one or more carbon atoms of the compound is ¹¹ C.
In some embodiments, one or more carbon atoms of the compound is ¹³ C.
In some embodiments, one or more carbon atoms of the compound is ¹⁴ C.
In some embodiments, one or more nitrogen atoms of the compound is ¹⁵ N.

In some embodiments, one or more or all one or more or all carbon atoms of the R ^3NA , R ^3NB , R ^7NA , R ^7NB , R ¹ , R ⁹ , R ^A and R ^B groups. Is ¹¹ C, ¹³ C or ¹⁴ C.
In some embodiments, one or more or all one or more or all carbon atoms of R ^3NA , R ^3NB , R ^7NA and R ^7NB are ¹¹ C, ¹³ C, or ¹⁴ C.

Combinations All compatible combinations of the above embodiments are explicitly disclosed herein as if each combination was specifically and individually described.

In particular, in the compounds of the present invention, the R ^3NA , R ^3NB , R ^7NA , R ^7NB , R ¹ , R ⁹ , R ^A and R ^B (and R ^AB ) groups are defined as independent variables, and those ^skilled in the art It will be understood that any compatible combination of these groups and substituents may be utilized in the compounds and methods of the present invention.

  Accordingly, all compatible combinations of these and other defined variables are expressly included in the present invention, as if each and all combinations were individually and explicitly described. Disclosed herein.

Some Preferred Embodiments In some embodiments, the compounds of the present invention can be selected from the following compounds, and pharmaceutically acceptable salts, solvates and hydrates thereof:

  One particular compound of the invention is the following compound 1:

  N, N, N ', N',-tetramethyl-10H-phenothiazine-3,7-diaminium bis (methanesulfonate).

This compound has the following:
N, N, N ′, N ′,-tetramethyl-10H-phenothiazine-3,7-diaminebis (hydromethanesulfonate)
Leucomethylthioninium bis (hydromethanesulfonate)
Leucomethylthioninium bis (mesylate)
LMTM
LMT. 2MsOH
Can also be called.

Purity The compounds of the present invention can conveniently be described as being “stabilized reduced forms”. The compound is oxidized (eg, auto-oxidized) to produce the corresponding oxidized form. Therefore, it may be inevitable that the composition containing the compound of the present invention contains at least a part of the corresponding oxidized compound as an impurity.

  Accordingly, another aspect of the present invention is herein a substantially purified form and / or a form that is substantially free of contaminants (eg, corresponding oxidized compounds, other contaminants). Relates to the described compounds.

  In some embodiments, the substantially purified form is at least 50% pure, such as at least 60% pure, such as at least 70% pure, such as at least 80% pure, such as at least 90% pure, For example, at least 95% purity, such as at least 97% purity, such as at least 98% purity, such as at least 99% purity.

  In some embodiments, the contaminants are 50% or less, such as 40% or less, such as 30% or less, such as 20% or less, such as 10% or less, such as 5% or less, such as 3% or less. For example, it corresponds to 2% by weight or less, for example, 1% by weight or less.

Product by Process In some embodiments, the compound is obtained or obtainable by the methods described herein.

Chemical Synthesis Methods for chemical synthesis of the compounds of the present invention are described herein. These and / or other well-known methods can be modified and / or adapted in a known manner to facilitate the synthesis of additional compounds within the scope of the present invention.

  Formula (I):

  The compound of the following formula (II):

{Where
R ¹ , R ⁹ , R ^3NA , R ^3NB , R ^7NA , and R ^7NB are as defined above}
Can be prepared from

  The compound of formula (II) is, for example, the following formula (III):

{Where
R ^Prot is an amine protecting group and R ¹ , R ⁹ , R ^3NA , R ^3NB , R ^7NA , R ^7NB , R ^A and R ^B are as defined above}
Can be prepared from

As a non-limiting example, R ^Prot can be an acyl group, such as an acetyl (—C (═O) Me) or benzyl (—C (═O) Ph) group.

  Compounds of formula (II) can be prepared, for example, by deprotection of compounds of formula (III) or by other known methods. Conversely, compounds of formula (II) can be generated by protection of compounds of formula (III).

  Compounds of formula (II) and (III) are known and can be prepared from known and / or commercially available starting materials, for example from the corresponding phenothiazine compounds, using known methods.

  For example, the intermediates of formulas (II) and (III) can be used for 3,7-diamino-10H-phenothiazine hydrochloride, hydrobromide, and iodination disclosed in PCT International Patent Application Publication No. WO2007 / 110627. Used in the process for the synthesis of hydrates.

  As disclosed in the above document, suitable phenothiazines can be converted to the corresponding 3,7-dinitro-phenothiazines using, for example, sodium nitrite and acetic acid and chloroform.

  The ring amino group can then be protected as the acetate salt using, for example, acetic anhydride and pyridine.

  The nitro group can then be reduced to an amino group using, for example, tin chloride II and ethanol.

  The amino group can then be substituted, eg, disubstituted, eg, methyl disubstituted, eg, methyl iodide, sodium hydroxide, DMSO, and tetra-n- Substitution with butylammonium bromide can provide N-acetyl protected 3,7-dialkylamino-10H-phenothiazine.

  Examples of such methods are illustrated in Schemes 1a and 1b. The use of any one or more of the reagents described herein in the method is of course encompassed by the present invention:

  The amino group of the N-acetyl intermediate can then be deprotected, ie the N-acetyl group can be removed using, for example, an aqueous acid.

Compounds of formula (II) and (III) can also be prepared using the methods disclosed in PCT International Patent Application Publication No. WO2008 / 007074. This document discloses compounds of formula (III) and compounds of formula (II), wherein R ^Prot is an acyl group, for example an acetyl group.

In one approach, first, for example, reaction with hydrazine (NH ₂ NH ₂ ), methyl hydrazine (MeNHNH ₂ ), or sodium borohydride (NaBH ₄ ); and acetic anhydride ((H ₃ CCO) ₂ O). By appropriate thioninium chloride in the presence of a suitable base such as, for example, pyridine (C ₆ H ₅ N) or Hunig's base (diisopropylethylamine, C ₈ H ₁₉ N), for example in a suitable solvent such as ethanol or acetonitrile. (Eg, methylthioninium chloride, ethylthioninium chloride, etc.) may be reduced and acetylated to yield the corresponding 1- (3,7-bis-dimethylamino-phenothiazin-10-yl) -ethanone. it can. The reduced and acetylated compound (of formula (III)) is then deprotected (by removing the acetyl group), for example by reaction with a suitable acid, to give the compound of formula (II) Can be generated or used directly. Advantageously, this reaction can produce a pure product.

  An example is shown in the following scheme.

  In another approach, for example, reaction with the reducing agents phenylhydrazine, ethanol, acetic anhydride and pyridine reduces the appropriate thioninium salt, such as ethyl thioninium semi zinc chloride, simultaneously, Ring amino groups can be protected.

An example is shown in the following scheme:

  Accordingly, in one aspect, the present invention provides the following formula (I):

  Of a 3,7-diamino-10H-phenothiazine compound of the following formula (II):

{Where
R ^A , R ^B , R ¹ , R ⁹ , R ^3NA , R ^3NB , R ^7NA , and R ^7NB are as defined above}
A process for the preparation from a compound of

  In some embodiments, the method includes the following: salt formation (SF) steps.

In some embodiments, salt formation (SF) comprises treatment of a compound of formula (II) with a suitable sulfonic acid.
In some embodiments, salt formation comprises treatment of a solution of a compound of formula (II) with a suitable sulfonic acid in an organic solvent.

  In a further aspect, the present invention provides the following formula (I):

  Of a 3,7-diamino-10H-phenothiazine compound of the following formula (III):

{Where
R ^A , R ^B , R ¹ , R ⁹ , R ^3NA , R ^3NB , R ^7NA , and R ^7NB are as defined above, and R ^Prot is an amine protecting group}
A process for the preparation from a compound of

A wide variety of amine protecting groups are widely used and well known in organic synthesis. For example, Protective Groups in Organic Synthesis (T Green and P. Wuts;. 4 th Edition; John Wiley and Sons, 2006) reference.

  In some embodiments, the amine protecting group is an acid cleavable protecting group.

  In some embodiments, the amine protecting group is an acyl group such as an acetyl group.

In some embodiments, the method comprises the following:
A ring amino deprotection (DP) step;
A salt formation (SF) step;
including.

Ring amino deprotection (DP) involves removal of a protecting group to convert an N-protected ring amine group (—NR ^Prot —) to a free ring amine group (—NH—). Deprotection of the compound of formula (III) produces the corresponding compound of formula (II).

Methods for the removal of amine protecting groups are known in the art. . For example, Protective Groups in Organic Synthesis (T Green and P. Wuts;. 4 th Edition; John Wiley and Sons, 2006) reference.

  In some embodiments, the ring amino deprotection (DP) step and the salt formation (SF) step are performed simultaneously (ie, as one step). For example:

  In some embodiments, simultaneous ring amino deprotection (DP) and salt formation (SF) can be performed with a suitable sulfonic acid of formula (III) to form a bis (sulfonate) salt of formula (I). Including treatment of compounds.

  In some embodiments, simultaneous ring amine deprotection and salt formation can include treatment of a solution of a compound of formula (III) in an organic solvent with sulfonic acid and water.

  In some embodiments, the organic solvent is toluene.

In the process of the present invention, the sulfonic acid can be selected from alkyl sulfonic acids and aryl sulfonic acids. It can be a sulfonic acid of formula R ^A SO ₃ H or R ^B SO ₃ H, where R ^A and R ^B are as defined herein.

  In some embodiments, the sulfonic acid can be a disulfonic acid, ie, a compound that includes two sulfonic acid moieties per molecule. These sulfonic acid moieties can be linked by an alkylene or arylene group.

  In some embodiments, the sulfonic acid is: methanesulfonic acid (MsOH), ethanesulfonic acid (EsOH), benzenesulfonic acid (BSA), naphthalenesulfonic acid (NSA), p-toluenesulfonic acid (TsOH) Ethanedisulfonic acid (EDSA), propanedisulfonic acid (PDSA) and naphthalene-1,5-disulfonic acid (NDSA).

  In some embodiments, the phenothiazine starting material (ie, the compound of formula (III)) is first heated in the organic solvent until completely dissolved, and the resulting solution is added to the reagents (ie, sulfonic acid and Filtered before addition of water).

  In some embodiments, the compound is heated in the organic solvent at a temperature of about 60-80 ° C, such as about 70 ° C.

  In some embodiments, the sulfonic acid is added in an amount of at least 2 molar equivalents, such as about 2.2 molar equivalents, relative to the phenothiazine starting material. It is understood that when disulfonic acid is used, the molar amount of acid will be at least 1 molar equivalent, for example about 1.1 molar equivalents, so as to achieve the same number of sulfonic acid moieties per molecule of phenothiazine starting material. Will be done.

  It may be desirable to add sulfonic acid slowly to prevent temperature rise (exotherm). Thus, in some embodiments, the sulfonic acid is added gradually. In some embodiments, the sulfonic acid is added at a temperature of about 15-25 ° C.

  In some embodiments, after the addition of sulfonic acid and water, the reaction is heated to a temperature of about 80-90 ° C. In some embodiments, the reaction is maintained at this temperature until determined to be complete, for example, by chromatographic analysis.

  In some embodiments, after the reaction, the solution is treated with a counter solvent to precipitate the product. In some embodiments, the counter solvent is an alcohol such as ethanol.

  It may be desirable to “seed” the reaction mixture with a small amount, eg, about 1 mg of the desired bis (sulfonate) product per gram of starting material (compound of formula (II)). Without wishing to be bound by theory, it is believed that seed addition ensures early and efficient precipitation of the desired product and reduces the chances of potential side reactions and by-product formation. It is done. Seeds are also believed to be effective in controlling the particle size of the precipitated product.

Thus, in some embodiments, after the reaction, the resulting mixture is seeded with a small amount of the desired bis (sulfonate) salt.
In some embodiments, the seed comprises the desired ground bis (sulfonate) salt.
In some embodiments, the seed comprises the desired bis (sulfonate) salt ground to a size of less than about 100 μm.

In some embodiments, the precipitated product is isolated by filtration.
In some embodiments, after filtration, the product is washed with an organic solvent such as ethanol or acetonitrile.

  Salt formation (SF) produces a bis (sulfonate) salt of formula (I) from a compound of formula (II):

  As explained above, bis (sulfonate) salts can also be prepared directly from the corresponding amino-protected (eg N-acetyl) compounds of formula (III).

  In this case, salt formation can be carried out simultaneously with deprotection, for example by using a suitable sulfonic acid such as methanesulfonic acid for the deprotection step. An example is illustrated in the following scheme:

  In a further aspect, the present invention provides the following formula (I):

{Where
R ^A , R ^B , R ¹ , R ⁹ , R ^3NA , R ^3NB , R ^7NA , and R ^7NB are as defined above}
A method for the preparation of a compound is provided, the method comprising:
Preparing a compound of formula (II) or (III) as defined herein;
Followed by a salt formation (SF) step and / or a cyclic amine deprotection (DP) step.

  The steps of salt formation (SF) and cyclic amine deprotection (DP) are as described above.

In some embodiments, preparing the compound of formula (II) or (III) above comprises the method described in PCT International Patent Application Publication No. WO2007 / 110627.
In some embodiments, preparing the compound of formula (II) or (III) above comprises the method described in PCT International Patent Application Publication No. WO2008 / 007074.

  In some embodiments, preparing the compound of formula (II) comprises ring amine deprotection (DP) of the compound of formula (III), as described above.

In some embodiments, preparing the compound of formula (III) comprises the following:
Nitriding (NO),
Ring amino protection (AP),
Nitrogen reduction (NR),
Amine substitution (AS)
One or more steps selected from:

In some embodiments, preparing the compound of formula (III) comprises the following:
A reduction (RED) step;
Ring amino protection (AP) step.

  The above steps may be performed in any logical order. In some embodiments, the steps are performed in the order listed (ie, any step in the list is performed at the same time as or after the previous step in the list).

In some embodiments, the nitridation (NO) is:
Nitriding (NO), where 10H-phenothiazine is converted to 3,7-dinitro-10H-phenothiazine, for example:

including.

  In some embodiments, nitridation is performed using a nitrite such as sodium nitrite, such as sodium nitrite and acetic acid, and a solvent such as dimethyl sulfoxide, dimethylformamide, acetonitrile, tetrahydrofuran, dimethoxyethane, acetone, dichloromethane or chloroform. Implemented.

In some embodiments, the ring amino protection (AP) is:
Ring amino protection (AP), where the ring amino group (—NH—) of 3,7-dinitro-10H-phenothiazine is converted to a protected ring amino group (—NR ^Prot ), for example:

  including.

  In some embodiments, ring amino protection is achieved as the acetate salt using, for example, acetic anhydride, eg, using a base such as acetic anhydride and an amine base, such as triethylamine or pyridine.

In some embodiments, the nitrogen reduction (NR) step includes the following:
Nitrogen reduction (NR), where each nitro (—NO ₂ ) group of protected 3,7-dinitro-10H-phenothiazine is converted to an amino (—NH ₂ ) group, for example: :

In some embodiments, the nitrogen reduction can be performed using, for example, tin chloride II, such as tin chloride II and ethanol. .
In some embodiments, the nitrogen reduction can be performed using palladium charcoal (Pd / C) and hydrogen, for example, in 2-methyl-tetrahydrofuran.
In some embodiments, the nitrogen reduction can be performed with zinc and aqueous ammonium chloride, for example, in methanol and THF.

In some embodiments, the amine substitution (AS) step is as follows:
Amine substitution (AS), where each amino (—NH ₂ ) group of the protected 3,7-diamino-10H-phenothiazine is converted to a disubstituted amino group, for example:

In some embodiments, the amine substitution is performed using an alkyl halide, such as an alkyl iodide, such as methyl iodide, such as methyl iodide and sodium hydroxide, DMSO, toluene, and tetra-n-butylammonium bromide. To be implemented.
In some embodiments, amine substitution includes treatment with formaldehyde (eg, paraformaldehyde, formalin) under reducing conditions. For example, treatment with formalin and hydrogen gas in the presence of a Pd / C catalyst; or treatment with paraformaldehyde in the presence of a reducing agent such as sodium cyanoborohydride and acetic acid.

In some embodiments, the reduction (RED) step includes the following:
Reduction (RED), where 3,7-di (disubstituted amino) -thioninium salt is a reducing agent such as, for example, hydrazine (NH ₂ NH ₂ ), methyl hydrazine (MeNHNH ₂ ), or sodium borohydride and Reduced by treatment with a base such as pyridine, triethylamine or Hunig's base (diisopropylethylamine) to give the corresponding 3,7-di (disubstituted amino) -10H-phenothiazine,
It is.

In some embodiments, the ring amino protection (AP) step comprises the following:
Ring amino protection (AP), where 3,7-di (disubstituted amino) -10H-phenothiazine is protected by, for example, treatment with acetic anhydride to give the corresponding protected 3,7-di (disubstituted Amino) -10H-phenothiazine, for example the corresponding N-acetyl 3,7-di (disubstituted amino) -10H-phenothiazine,
It is.

  In some embodiments, the above steps are performed in the order listed (ie, any step in the list is performed at the same time as or after the preceding step in the list).

  In some embodiments, the reduction (RED) step and the ring amino protection (AP) step are performed simultaneously (ie, as one step).

For example, in some embodiments, the combined reduction (RED) step and ring amino protection (AP) step include the following:
Reduction (RED) and ring amino protection (AP), wherein the 3,7-di (disubstituted amino) -thioninium salt is reduced to give the corresponding 3,7-di (disubstituted amino) -10H-phenothiazine. And the 3,7-di (disubstituted amino) -10H-phenothiazine ring amino group (—NH—) is converted to a protected ring amino group (—R ^Prot ) to give the corresponding protected 3 , 7-di (disubstituted amino) -10H-phenothiazine, for example as follows:

{Wherein Y is a counter ion}
In some embodiments, Y represents Cl.

  In some embodiments, the 3,7-di (disubstituted amino) -thioninium salt is methylthioninium chloride (MTC).

In some embodiments, the combined reduction (RED) step and ring amino protection (AP) step comprises phenylhydrazine, MeNHNH ₂ , or NH ₂ NH ₂ . Achieved with hydrazine such as H ₂ O and acetic anhydride.

  In some embodiments, the above steps are performed under a nitrogen atmosphere.

  In some embodiments, the combined reduction (RED) step and ring amino protection (AP) step are performed using, for example, phenylhydrazine, ethanol, acetic anhydride and pyridine.

  In some embodiments, the combined reduction (RED) step and ring amino protection (AP) step are performed under a nitrogen atmosphere using, for example, hydrazine hydrate, acetonitrile, acetic anhydride, and triethylamine.

In some embodiments, the protected 3,7-di (disubstituted amino) -10H-phenothiazine, eg, N-acetyl 3,7-di (disubstituted amino) -10H-phenothiazine undergoes a purification step. .
In some embodiments, purification includes the addition of an organic solvent such as toluene and an acid such as acetic acid to dissolve the compound, followed by a washing step.
In some embodiments, washing comprises adding water and / or aqueous acetic acid to the compound solution; stirring and / or heating; and separating the organic layer.
In some embodiments, the washing is repeated, for example, up to 3 times.
In some embodiments, the isolation of the purified product follows the wash.
In some embodiments, the isolation of the purified product comprises cooling the product,
Including precipitation and filtration.

Crystalline Forms In some embodiments, the compounds of the invention are provided in crystalline form.
In some embodiments, the crystalline form is “Form A” as described herein.
In some embodiments, the crystalline form has the structure shown in FIG. 17 and / or the crystallographic data shown in Appendix Table 1 and / or the atomic coordinates shown in Appendix Table 2 and / or Or the bond length and bond angle shown in Appendix Table 3 and / or the anisotropic displacement parameter shown in Appendix Table 4 and / or the hydrogen coordinate and isotropic displacement parameter shown in Appendix Table 5 Features.

Reversal and / or Inhibition of Protein Aggregation One aspect of the present invention is for modulating (eg, reversing and / or inhibiting) protein aggregation, eg, protein aggregation associated with neurodegenerative diseases and / or clinical dementia. Or the use of a compound or composition as described herein. Aggregation may be in vitro or in vivo and may be related to the pathology discussed below.

  Accordingly, one aspect of the invention is a method of modulating (eg, reversing and / or inhibiting) protein aggregation, eg, protein aggregation associated with neurodegenerative disease and / or clinical dementia. Wherein said protein comprises contacting said protein with an effective amount of a compound or composition described herein. The method can be performed in vitro or in vivo.

  Similarly, one aspect of the invention pertains to methods of modulating (eg, reversing and / or inhibiting) protein aggregation in a mammalian brain, wherein the aggregation is associated with the pathology described herein. Wherein said treatment comprises administering to said mammal in need thereof a prophylactically or therapeutically effective amount of a compound or composition described herein that is an inhibitor of said aggregation.

Method of treatment Another aspect of the invention comprises administering to a patient in need of treatment a prophylactically or therapeutically effective amount of a compound described herein, preferably in the form of a pharmaceutical composition. It is related with the method of treatment including.

Use in therapeutic methods Another aspect of the invention pertains to compounds or compositions described herein for use in a method of treatment of the human or animal body (eg, for a disease state) by therapy.

Use in the manufacture of a medicament Another aspect of the present invention relates to the use of a compound or composition described herein in the manufacture of a medicament for use in the treatment (eg of a disease state).

  In some embodiments, the medicament comprises a compound of the invention.

  In some embodiments, the medicament is a composition as described below.

Treated Disease Conditions—Protein Aggregating Diseases The compounds and compositions of the invention are useful in the treatment or prevention of protein aggregating diseases.

  Thus, in some embodiments, the disease state is a protein-aggregating disease, eg, the treatment is described in an amount sufficient herein to inhibit aggregation of proteins associated with the disease state. Depending on the compound or composition.

  In general, protein aggregation is due to induced structural polymerization interactions, i.e. those in which structural changes in the protein or fragments thereof cause self-proliferative templated binding and aggregation of additional (precursor) protein molecules. to cause. When nucleation begins, an aggregation cascade follows, which involves induced structural polymerization of additional protein molecules and is a toxic product in the aggregate that is substantially resistant to further proteolysis. Leads to the formation of fragments. The protein aggregates thus formed are thought to be a major cause of pathological manifestations as neurodegenerative diseases, clinical dementia, and other pathological symptoms.

  The following table lists the aggregating proteins associated with various diseases and the corresponding protein aggregating diseases. The use of the compounds and compositions of the present invention for these proteins or diseases is encompassed by the present invention.

  As described in PCT International Patent Application Publication Nos. WO02 / 055720, WO2007 / 110630, and WO2007 / 110627, diaminophenothiazine is useful in the prevention of such protein-aggregating diseases.

  Thus, unless otherwise required by context, descriptions of embodiments relating to tau protein or tau-like protein (eg, MAP2; see below) may refer to other proteins (eg, β-amyloid, synuclein) discussed herein. , Prions, etc.), or other proteins that initiate or suffer similar pathological aggregation by conformational changes in regions important in the propagation of this aggregation, or confer proteolytic stability to aggregates thus formed Should be construed as equally applicable (eg "Neurobiology of Alzheimer's Disease", 2nd Edition, 2000, Eds. Dawbarn, D. and Allen, SJ, The Molecular and Cellular Neurobiology Series, Bios (See the article by Wischik et al. In Scientific Publishers, Oxford). All such proteins can be referred to herein as “aggregating disease proteins”.

  Similarly, where reference is made herein to “tau-tau aggregation,” etc., this should be construed to apply to other “aggregating protein aggregations” such as β-amyloid aggregation, prion aggregation, synuclein aggregation, etc. Can do. The same applies to “tau proteolysis”.

Preferred Aggregating Disease Proteins A preferred embodiment of the invention is based on tau protein. As used herein, the term “tau protein” generally refers to any protein of the tau protein family. Tau protein has been characterized as one of a larger family of proteins that co-purify with microtubules during repeated construction and degradation cycles (eg, Shelanski et al., 1973, Proc. Natl. Acad. Sci USA, Vol. 70, pp. 765-768), known as microtubule-associated proteins (MAPs). Members of the tau family consist of a characteristic N-terminal segment, a sequence of about 50 amino acids inserted in the N-terminal segment that is developmentally regulated in the brain, a characteristic consisting of 3 or 4 tandem repeats of 31-32 amino acids Share the common feature of having a typical tandem repeat region and a C-terminus.

  MAP2 is a major microtubule-associated protein in the soma dendritic compartment (eg, Matus, A., in “Microtubules” [Hyams and Lloyd, Eds.] Pp. 155-166, John Wiley and Sons, New (See York, USA). The MAP2 isoform is almost identical to the tau protein within the tandem repeat region, but differs substantially in both the sequence and range of the N-terminal domain (eg, Kindler and Garner, 1994, Mol. Brain Res., Vol. 26, pp. 218-224). Nevertheless, aggregation in the tandem repeat region is not selective for the tau repeat domain. Thus, it will be understood that any discussion relating to tau protein or tau-tau aggregation herein should be construed as also relating to tau-MAP2 aggregation, MAP2-MAP2 aggregation, and the like.

  In some embodiments, the protein is a tau protein.

  In some embodiments, the protein is synuclein, eg, α- or β-synuclein.

  In some embodiments, the protein is TDP-43.

  TAR DNA binding protein 43 (TDP-43) is a 414 amino acid protein encoded by TARDBP on chromosome 1p36.2. This protein is highly conserved, widely expressed, and mainly localized in the nucleus, but can travel between the nucleus and the cytoplasm (Mackenzie et al 2010). It is involved in transcription and splicing regulation and may also have a role in other processes such as microRNA processing, apoptosis, cell division, messenger RNA stabilization, regulation of neuroplasticity and maintenance of dendritic integrity. Furthermore, since 2006, a considerable amount of evidence has accumulated to support the functional hypothesis TDP-43 toxicity increase in amyotrophic lateral sclerosis (ALS). TDP-43 is an intrinsically prone protein and aggregates formed in vitro are ultrastructurally similar to TDP-43 deposits found in degenerative neurons in ALS patients (Johnson et al 2009). Johnson et al. (2008) showed that when TDP-43 is overexpressed in a yeast model, only the aggregated form is toxic. Several in vitro studies have also shown that the C-terminal fragment of TDP-43 is more likely to ubiquitinate and form insoluble cytoplasmic aggregates that are toxic to cells than full-length TDP-43. (Arai et al 2010; Igaz et al2009; Nonaka et al 2009; Zhang et al 2009). Despite Nonaka et al. (2009) suggesting that these cytoplasmic aggregates bind to and deplete the endogenous full-length protein from the nucleus, Zhang et al. (2009) maintain normal nuclear expression. And suggested a pure toxic effect on the aggregates. Yang et al. (2010) described that full-length TDP-43 was trapped within aggregates of C- and N-terminal fragments of TDP-43 in cultured NSC34 motoneurons. Impaired neurite outgrowth as a result of the presence of such truncated fragments can be rescued by overexpression of the full-length protein. Although the role of neurite outgrowth in vivo has not yet been confirmed, this model will support the suggestion by Nonaka et al. Regarding the role of TDP-43 in ALS pathogenesis.

  Expression of TDP-43 mutants in cell culture has been repeatedly reported to result in increased production of C-terminal fragments and greater cytoplasmic aggregation and toxic effects than wild-type proteins (Kabashi et al 2008; Sreedharanet al 2008; Johnson et al 2009; Nonaka et al 2009; Arai et al 2010; Barmarda etal 2010; Kabashi et al 2010).

  In some embodiments of the invention, where the protein is a tau protein (eg, optionally in the form of paired helical fibrils (PHFs) in neurofibrillary tangles (NFTs) in the mammalian brain). A method of inhibiting the production of protein aggregates is provided wherein the treatment is as described above.

Preferred indications-protein aggregating diseases In particular, it is not only Alzheimer's disease (AD) that tau protein (and its abnormal function or processing) can play a role. The pathogenesis of neurodegenerative disorders such as Pick's disease and progressive supranuclear palsy (PSP) is associated with the accumulation of pathologically truncated tau aggregates in the dentate gyrus and neocortical astrocytes, respectively. It seems to be. Other dementias are frontotemporal dementia (FTD); FTD with parkinsonism linked to chromosome 17 (FTDP-17); desuppression / dementia / parkinsonism / muscular atrophy (DDPAC); Paleoblastic substantia nigra degeneration (PPND); Guam ALS syndrome; Paleoblastic subtilis degeneration (PNLD); Cortical basal ganglia degeneration (CBD) and the like (for example, “Neurobiology of Alzheimer's Disease”, 2nd Edition, 2000, Eds. Dawbarn, D. and Allen, SJ, The Molecular and Cellular Neurobiology Series, Bios Scientific Publishers, Oxford, in an article by Wischik et al; see particularly Table 5.1). All of these diseases characterized primarily or in part by abnormal tau aggregation are referred to herein as “tauopathy”.

Thus, in some embodiments, the disease state is tauopathy.
In some embodiments, the disease state is neurodegenerative tauopathy.

  In some embodiments, the disease state involves Alzheimer's disease (AD), Pick's disease, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), parkinsonism linked to chromosome 17. FTD (FTDP-17), frontotemporal lobar degeneration (FTLD) syndrome, disinhibition / dementia / parkinsonism / muscular atrophy complex (DDPAC), pallidum bridge substantia nigra degeneration (PPND), Guam ALS syndrome, Paleoblastic lupus degeneration (PNLD), cerebral cortex basal ganglia degeneration (CBD), taste-granular dementia (AgD), boxer dementia (DP) or chronic traumatic brain injury (CTE), down Syndrome (DS), Lewy body dementia (DLB), subacute sclerosing panencephalitis (SSPE), MCI, C type Niemann-Pick disease (NPC), type B Sanfilip syndrome (mucopolysaccharidosis II) B), or myotonic dystrophy (DM), selected from DM1 or DM2 or chronic traumatic brain injury, (CTE).

  In some embodiments, the disease state is lysosomal storage disease with tau lesions. NPCs are caused by mutations in the NPC1 gene that affect cholesterol metabolism (Love et al 1995), and type B Sanfilipo syndrome is caused by mutations in the NAGLU gene, accumulating heparin sulfate in the lysosome (Ohmi). et al. 2009). In these lysosomal storage diseases, tau lesions are observed and the treatment can reduce disease progression. Other lysosomal storage diseases may also be characterized by tau accumulation.

  The use of phenothiazinediaminium in the treatment of Parkinson's disease and MCI is described in more detail in PCT international patent applications PCT / GB2007 / 001105 and PCT / GB2008 / 002066.

  In some embodiments, the disease state is Parkinson's disease, MCI, or Alzheimer's disease.

In some embodiments, the disease state is other such as Huntington's disease or bulbospinal muscular atrophy (or Kennedy's disease), and dentate nucleus red nucleus pallidal atrophy, and various spinocerebellar ataxias Of polyglutamine disease.
In some embodiments, the disease state is FTLD syndrome (see, eg, may be tauopathy or TDP-43 proteinopathy, see below).
In some embodiments, the disease state is PSP or ALS.

  In some embodiments, the treatment (eg, treatment of neurodegenerative tauopathy, eg, treatment of Alzheimer's disease) optionally includes one or more other agents, eg, one or more ((Acipept ™) Donepezil (also known as Exelon ™), rivastigmine (also known as Exelon ™), cholinesterase inhibitors (such as Galantamine (also known as Reminyl ™)), (also as (Ebixa ™, Namenda ™) It may be used in combination with (known) NMDA receptor antagonists (such as memantine), muscarinic receptor agonists, and / or inhibitors of amyloid precursor protein processing leading to enhanced production of β-amyloid.

  TDP-43 proteinopathy includes amyotrophic lateral sclerosis (ALS; ALS-TDP) and frontotemporal lobar degeneration (FTLD-TDP).

  The role of TDP-43 in neurodegeneration in ALS and other neurodegenerative disorders has been reviewed in several recent publications (Chen-Plotkin et al 2010; Gendron et al 2010; Geser et al 2010; Mackenzie et al 2010).

  ALS is characterized by progressive paralysis and muscle wasting as a result of degeneration of both upper and lower motor neurons in the primary motor cortex, brainstem and spinal cord. It is sometimes referred to as motor neuron disease (MND), but there are other diseases besides ALS that affect either upper or lower motor neurons. A definitive diagnosis requires clear evidence of clinical progression that cannot be explained by signs of both upper and lower motor neurons in the medulla, arm and leg musculature and the course of any other disease (Wijesekera and Leigh 2009) .

  Despite the majority of cases being ALS-TDP, there are other cases where the pathological protein differs from TDP-43. Misfolded SOD1 is a pathogenic protein in ubiquitin-positive inclusion bodies in ALS with mutations in SOD1 (Seetharaman et al 2009), a very small subset of familial ALS (approximately 3 In ~ 4%), the pathogenic protein ubiquitinated by mutation in FUS (fused into sarcoma protein) is FUS (Vance et al 2009; Blair et al 2010) impaired nuclear translocation of FUS Although the means are still unclear, like TDP-43, FUS appears to be important in nucleocytoplasmic shuttling. The new molecular level classification of ALS, modified from Mackenzie et al. (2010), reflects distinct underlying pathological canisism in various subtypes (see table below).

  New molecular level classification of ALS (modified from Mackenzie et al., 2010). In the majority of cases, TDP-43 is a pathological ubiquitinated protein found in ALS.

  Amyotrophic lateral sclerosis has been recognized as a disease classification entity for nearly a century and a half and is classified as a subtype of MND in ICD 10 (G12.2). A reliable clinical diagnosis, slightly different from Charcot's original description, is available for ALS, and neuropathological criteria that reflect the underlying molecular pathology have also been agreed.

  ALS is pathologically classified into three subgroups, ALS-TDP, ALS-SOD1 and ALS-FUS, but the last two conditions are rare. The largest trial to date has shown that all sporadic ALS cases have TDP-43 pathology (Mackenzie et al 2007). Only about 5% of ALS is familial (Byrne et al 2010), SOD1 is mutated, the most common mutation is found in FALS, accounting for 12-23% of cases (Andersen et al 2006) ). SOD1 may also be involved in 2-7% of SALS. Mutations in FUS are much rarer, only about 3-4% of FALS (Blair et al 2010). Therefore, it can be reliably predicted that clinical cases of SALS will have a pathology based on TDP-43. Similarly, mutations in TDP-43, which account for about 4% of cases, are reliably predictable in FALS (Mackenzie et al 2010). ALS with mutations in VCP (Johnson et al 2010), ANG (Seilhean et al 2009), and CHMP2B (Cox et al 2010), accounting for 1-2% of FALS, is also associated with TDP-43 positive pathology It has been reported. Although mutations in SOD1, FUS and ATXN2 have not been found to be associated with TDP-43 positive aggregates, TDP-43 has been reported to be involved in pathological processes presumed to be due to these mutations (Higashi et al 2010; Ling et al2010; Elden et al 2010).

  Thus, it was established that TDP-43 has an important and potential central role in the overwhelming majority of etiology of SALS cases and may be involved in a significant proportion of the pathogenesis of FALS. Currently, ALS is widely considered to be a TDP-43 proteinopathy (Neumann et al 2009), and numerous in vitro and in vivo studies have shown that toxic gain of function by aggregation of TDP-43 It supports the hypothesis that it contributes to at least some of the neurotoxicity of the disease.

  FTLD syndrome is an insidious progressive neurodegenerative condition with an insidious onset that peaks in late middle age. First-degree relatives often have a similar family history of similar disabilities.

  Behavioral FTD is characterized by early significant changes in social and interpersonal functions, often accompanied by repetitive behaviors and changes in dietary patterns. In semantic dementia, rephrasing difficulties are prominent despite otherwise verbal fluency, which is accompanied by a decrease in object knowledge and a difficulty in understanding one word in recognition evaluation. Progressive non-fluent aphasia presents a combination of speech movement problems and grammatical disorders. The clinical diagnostic features for these three FTLD syndromes are shown in the following table and all criteria of Neary et al. (1998).

  Immediately after the discovery that TDP-43 positive inclusion bodies characterize ALS and FTLD-TDP (Neumann et al 2006), a missense mutation in the TARDBP gene was confirmed in familial and sporadic cases of ALS ( Gitcho et al 2008; Sreedharanet al 2008). So far, 38 different TARDBP mutations have been reported in 79 genetically unrelated families worldwide (Mackenzie et al 2010). TARDBP mutations account for about 4% of all familial ALS cases and about 1.5% of sporadic ALS cases.

  As of December 2010, 13 gene mutations associated with familial and sporadic ALS have been identified. Although the association of ALS to the other five chromosomal loci has been demonstrated, no specific mutations have been identified so far.

Methylthioninium (MT) in TDP-43 proteinopathy
MT has an overwhelming number of familial and sporadic ALS pathologic features, and has a mode of action that can target and reduce intracellular TDP-43 protein aggregation, which is also a feature of FTLD-P.

  In addition, laboratory data indicate that methylthioninium inhibits the formation of TDP-43 aggregates in SH-SY5Y cells. After treatment with 0.05 μM MT, TDP-43 aggregates were reduced by 50%. These findings were confirmed by immunoblot analysis (Yamashita et al 2009).

  Thus, the compounds and compositions of the present invention can be useful for the treatment of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD).

Methylthioninium (MT) in Huntington's disease and polyglutamine disease
MT can reduce intracellular polyglutamine protein aggregation, a pathological feature of Huntington's disease. Huntington's disease is caused by expansion of the translated CAG repeat located at the N-terminus of huntingtin. Wild-type chromosomes contain 6-34 repeats, whereas in Huntington's disease the chromosome contains 36-121 repeats. The age of onset of the disease is inversely related to the length of the CAG zone that encodes a polyglutamine repeat in the protein.

  Laboratory data show that methylthioninium inhibits the formation of aggregates of huntingtin derivatives containing a stretch of 102 residues of polyglutamine (van Bebber et al. 2010). When tested at 0, 10 and 100 μM, MT prevented the formation of such aggregates in zebrafish in a dose dependent manner.

  Accordingly, the compounds and compositions of the present invention are useful in other applications such as Huntington's disease and bulbar spinal muscular atrophy (or Kennedy's disease), and erythrocytic red nuclei pallidal atrophy, and various spinocerebellar ataxias. Can be useful for the treatment of polyglutamine disease (Orr & Zoghbi, 2007).

The most frequently affected organ in mitochondrial and rafora mitochondrial disorders, particularly respiratory chain diseases (RCDs), is the central nervous system (CNS) in addition to skeletal muscle. CNS signs of RCDs include stroke-like episodes, epilepsy, migraine, ataxia, spasticity, movement disorders, mental disorders, cognitive decline, or even dementia (mitochondrial dementia). So far, mitochondrial dementia has been reported in MELAS, MERRF, LHON, CPEO, KSS, MNGIE, NARP, Ray syndrome, and Alpers-Futtenlocher disease (Finsterer, 2009). There are four complexes in the mitochondrial respiratory chain that are involved in a series of electron transfers. Abnormal function of any of these complexes leads to mitochondrial disease, followed by an abnormal electron transport chain and subsequent abnormal mitochondrial respiration. Mitochondrial respiratory chain complex III transfers electrons to cytochrome c.

  The compounds and compositions of the invention can also be used to treat mitochondrial diseases associated with deficiencies and / or disorders of complex III function of the respiratory chain. The above compounds have the ability to act as an effective electron carrier because of the low redox potential at which the thioninium moiety converts between oxidized and reduced forms. In the case of impaired and / or deficient complex III function leading to mitochondrial disease, the compounds of the present invention may be responsible for complex III electron transport and transfer due to the ability of the thioninium moiety to travel between oxidized and reduced forms. It can also play a role, thus acting as an electron carrier in place of suboptimally functioning complex III, transferring electrons to cytochrome c.

  The compounds and compositions of the present invention divert misfolded protein / amino acid monomers / oligomers from the accumulation and / or refolding pathway of Hsp70 ADP-related proteins, and convert these abnormally folded protein monomers / oligomers into these Produces an active thioninium moiety with the ability to redirect the Hsp70 ATP-dependent ubiquitin-proteasome system (UPS) directly through a pathway that removes misfolded protein / amino acid monomers / oligomers via the direct pathway It also has the ability (Jinwal et al 2009).

  Lafora disease (LD) is an autosomal recessive fatal epilepsy associated with the gradual accumulation of insoluble glycogen, called polyglycosan, in many tissues. In the brain, polyglucosan bodies or lafora bodies form in neurons. Inhibition of Hsp70 ATPase by MT (Jinwal et al. 2009) can up-regulate the removal of misfolded proteins. Lafora disease mainly results from inclusion bodies that can accelerate the aggregation of misfolded tau proteins, both of which are lysosomal due to mutations in either the Laforin or Malin genes located on chromosome 6. Due to a deficiency in the ubiquitin-proteasome system (UPS). Secondary mitochondrial damage by impaired UPS further leads to suppression of mitochondrial activity and disruption of the electron transport chain, resulting in additional lipofuscin and causing seizures characteristic of Lafora disease.

  The MT moiety can degrade existing tau aggregates by inhibiting Hsp70 ATPase, reducing further tau accumulation and increasing lysosomal efficiency. MT can lead to a reduction in tau entanglement by promoting the removal of tau monomers / oligomers by the ubiquitin proteasome system through its inhibitory action on Hsp70 ATPase.

  Thus, the compounds and compositions of the invention can be useful in the treatment of Lafora's disease.

Treated Disease Conditions—Other Disease Conditions In some embodiments, the disease condition is skin cancer.
In some embodiments, the disease state is melanoma.
In some embodiments, the disease state is a viral, bacterial or protozoal disease state.
In some embodiments, the (protozoan) disease state is malaria. The treatment may be combined with one or more antimicrobial agents, such as chloroquine and / or atobacon.

  In some embodiments, the (viral) disease state is caused by hepatitis C, HIV or West Nile virus (WNV).

Other Uses Another aspect of the present invention is a method for inactivating pathogens in a sample (eg, a blood or plasma sample) comprising introducing a compound described herein into the sample, and It relates to the use of said compounds in a method comprising a step of exposing to light.

  For example, in some embodiments, the method includes introducing the compound into a sample and then exposing the sample to light.

Use as ligands Compounds described herein that are capable of inhibiting aggregation of tau protein would also be able to act as ligands or labels for tau protein (or aggregated tau protein). Thus, in some embodiments, the compounds of the invention are ligands for tau protein (or aggregated tau protein).

  Such compounds (ligands) are stable and unstable detectable isotopes, radioisotopes, positron emitting atoms, magnetic resonance labels, dyes, fluorescent markers, antigenic groups, therapeutic moieties, or prognostic, diagnostic or therapeutic applications It can incorporate, conjugate, chelate, or otherwise bind to a chemical group, such as any other moiety that can aid.

  For example, in some embodiments, the compound is as defined herein, but the compound is one or more (eg, one, two, three, four, etc.) detectable. Incorporates, conjugates to, chelates with, or otherwise with a label, eg, an isotope, radioisotope, positron emitting atom, magnetic resonance label, dye, fluorescent marker, antigenic group or therapeutic moiety With the additional limitation of binding.

  In some embodiments, the compound is a ligand as well as a label, eg, a tau protein (or aggregated tau protein), and one or more (eg, one, two, three, four, etc.) Incorporating, conjugating to, chelating with or otherwise binding to a detectable label of

  For example, in some embodiments, the compound is as defined above, but the compound incorporates one or more (eg, one, two, three, four, etc.) detectable labels. With further limitations of conjugating to it, chelating with it, or otherwise binding to it.

  The labeled compound (eg, when linked to tau protein or aggregated tau protein) can be visualized or detected by any suitable means, and the skilled artisan will be able to use any suitable means known in the art. It will be appreciated that detection means can be used.

For example, the compound (ligand-label) incorporates a positron emitting atom (eg, ¹¹ C) (eg, as one or more alkyl group substituents, eg, a carbon atom of a methyl group substituent) It can be suitably detected by detecting the compound using known positron emission tomography (PET).

Such ¹¹ C-labeled compounds can be adapted, for example, from PCT International Patent Application Publication No. WO 02/075318 (see FIGS. 11a, 11b, 12 thereof) by adapting the methods described herein in a known manner. And can be prepared in the same manner as described in WO2005 / 030676.

  Accordingly, another aspect of the present invention is a method for labeling a tau protein (or aggregated tau protein), which comprises: (i) one or more (eg, 1 Two, three, four, etc.), or conjugated to, chelated with, or otherwise contacted with a compound that binds to it. The compound may be provided as a composition described herein.

  Another aspect of the present invention is a method for detecting tau protein (or aggregated tau protein), which comprises: (i) one or more (eg, one, tau protein (or aggregated tau protein)): (Ii) tau protein, incorporating, conjugated to, chelated with, or otherwise chelated with, or otherwise binds to, a detectable label of (2, 3, 4, etc.) Detecting the presence and / or amount of the compound bound to (or aggregated tau protein). The compound may be provided as a composition described herein.

  Another aspect of the present invention is a method for diagnosing or prognosing tau proteinopathy in a subject suspected of having a disease, comprising the following: (i) tau protein or aggregated tau protein, particularly tau protein Incorporate, conjugated to, or chelate with a detectable label of a compound capable of labeling (eg, one or more (eg, one, two, three, four, etc.) (Ii) a compound that otherwise binds to the subject; (ii) determining the presence and / or amount of the compound bound to tau protein or aggregated protein in the brain of the subject; (iii) The method includes the step of associating a determination result obtained in (ii) with a disease state of a subject. The compound may be provided as a composition described herein.

  Another aspect of the invention is a compound capable of labeling tau protein or aggregated tau protein (eg, one or more (eg, one, two, etc.) for use in a method for diagnosis or prognosis of tau proteinopathy. One, three, four, etc.) which incorporates, conjugates to, chelates with, or chelates with, or otherwise binds to it). The compound may be provided as a composition described herein.

  Another aspect of the present invention is a compound capable of labeling tau protein or aggregated tau protein, particularly tau protein in a method for producing a diagnostic or prognostic reagent for use in diagnosis or prognosis of tau proteinopathy (eg, tau protein) A compound that incorporates, conjugates to, chelates with, or otherwise binds to one or more (eg, one, two, three, four, etc.) detectable labels ) Regarding the use of. The compound may be provided as a composition described herein.

  Those skilled in the art will recognize that, instead of directly administering the ligand / label, they are present in the same subject as precursor forms that are converted to the active form (eg, linked form, labeled form) by an activator present or administered. It will be understood that can be administered.

  The ligands disclosed herein can be used as part of a diagnostic or prognostic method. It can be used to select a patient for treatment or to assess the effectiveness of a treatment or therapeutic agent (eg, an inhibitor of tau protein aggregation) administered to a subject.

The term “treatment” as used herein in relation to treatment of a treatment condition generally refers to treatments and therapies that achieve some desired therapeutic effect, whether in humans or animals (eg, in veterinary applications) For example, with respect to inhibiting the progression of a condition, it includes a decrease in progression rate, a halt in progression rate, a regression of the condition, a remission of the condition, and a cure of the condition. Treatment as a prophylactic measure (eg, prevention, prevention) is also included.

  The term “therapeutically effective amount” as used herein is an effective term that, when administered according to a desired treatment regimen, is effective to produce any desired therapeutic effect commensurate with a reasonable benefit / risk ratio. It relates to a compound of the invention, or its amount of material, composition or dosage form containing the compound.

  Similarly, the term “prophylactically effective amount” as used herein is effective to produce any desired prophylactic effect commensurate with a reasonable benefit / risk ratio when administered according to a desired treatment regimen. , Or the amount of a compound of the invention, or a material, composition or dosage form containing the compound.

  In light of the present description, the term “prevention” should not be understood as being limited to complete success, ie complete protection or complete prevention. In this context, prevention rather means a means administered prior to detection of a symptomatic condition for the purpose of maintaining health by helping to delay, relieve or avoid a particular condition.

  The term “treatment” includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. As examples of treatment and therapy, administration of active agents including chemotherapy (eg, drugs, antibodies (eg, in immunotherapy), prodrugs (eg, in photodynamic therapy, GDEPT, ADEPT, etc.)) ); Surgery; radiation therapy; and gene therapy.

  For example, it may be beneficial to combine treatment with a compound described herein with one or more other (eg, one, two, three, four) agents or therapies .

  The specific combination will be at his / her general knowledge and the discretion of the physician who will select the dose according to the dosing regimen known to the skilled practitioner.

  The agents (ie, the compounds described herein and one or more other agents) can be administered simultaneously or sequentially, and are administered via different routes, individually on different dosing schedules. be able to. For example, when administered continuously, the agent may be spaced at close intervals (eg, over a period of 5-10 minutes) or at longer intervals (eg, 1, 2, 3, 4 hours or more). (If necessary, more time) can be administered, and the exact dosing regimen is commensurate with the characteristics of the therapeutic agent.

  An agent (ie, a compound described herein and one or more other agents) can be formulated together in a single dosage form, or each agent can be formulated separately, Optionally, they can be provided together in the form of a kit with instructions for their use.

Route of Administration A compound of the invention or pharmaceutical composition comprising it can be administered to a subject / patient by any convenient route of administration, whether systemic / peripheral or local (ie, the desired site of action).

  Administration routes include: oral (eg, by ingestion); buccal; sublingual; transdermal (including, for example, by patch, plaster, etc.); transmucosal (including, for example, by patch, plaster, etc.); Intranasal (eg, via nasal drops); intraocular (eg, via eye drops); intrapulmonary (eg, via aerosol, eg, via mouth or nose, eg, by inhalation or gas infusion therapy); (eg, via suppositories or enemas) ) In the rectum; in the vagina (eg by pessary); for example, subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intrathecal, intracapsular, intracapsular, intraorbital, intraperitoneal, Parenteral by injection, including intratracheal, subepidermal, intraarticular, intrathecal and intrasternal injection (eg, including intravascular catheter injection into the brain); eg, subcutaneous or intramuscular depot or reservoir Include the embedding, but is not limited thereto.

  A preferred composition is an oral composition formulated as described in more detail below.

Subjects / patients Subjects / patients include animals, mammals, placental mammals, rodents (eg, guinea pigs, hamsters, rats, mice), mice (eg, mice), rabbits (eg, rabbits), birds (Eg, birds), dogs (eg, dogs), cats (eg, cats), horses (eg, horses), pigs (eg, pigs), sheep (eg, sheep), cows (eg, , Cows), primates, monkeys (eg monkeys or apes), monkeys (eg marmoset, baboon), single holes (eg platypus), apes (eg gorilla, chimpanzee, orangutan, gibbon) or human It may be.

  Furthermore, the subject / patient may be in any of its developmental forms, eg fetuses.

  In some embodiments, the subject / patient is a human.

Compositions / Formulations While the compounds of the invention can be used (eg, administered) alone, it is often preferable to provide them as a composition or formulation.

  Accordingly, another aspect of the present invention provides a composition comprising a compound described herein and a pharmaceutically acceptable carrier or diluent.

  In some embodiments, the composition comprises a pharmaceutical composition (eg, formulation, preparation) comprising a compound described herein and a pharmaceutically acceptable carrier, diluent or excipient. , Pharmaceutical).

  In some embodiments, the composition comprises at least one compound described herein as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, filler, buffering agent, preservative, antiseptic, One well known to those skilled in the art, including but not limited to oxidizing agents, lubricants, stabilizers, solubilizers, surfactants (eg, wetting agents), masking agents, coloring agents, flavoring agents and sweetening agents. Or a pharmaceutical composition comprising a plurality of other pharmaceutically acceptable ingredients.

  In some embodiments, the composition further comprises other active agents, such as other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients and the like can be found in standard pharmaceutical texts. For example, Handbook of Pharmaceutical Additives , 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences , 20th edition, pub.Lippincott, Williams & See Wilkins, 2000; and Handbook of Pharmaceutical Excipients , 2nd edition, 1994.

Another aspect of the invention relates to at least one [ ¹¹ C] -radiolabeled compound as defined herein, one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, for example The present invention relates to a method for producing a pharmaceutical composition comprising the step of mixing with a carrier, diluent, excipient and the like. When formulated as separate units (eg, tablets), each unit contains a predetermined amount (dose) of the compound.

  As used herein, the term “pharmaceutically acceptable” refers to the subject in question without an excessive toxicity, irritation, allergic reaction or other problem or complication, commensurate with a reasonable benefit / risk ratio ( For example, it relates to compounds, ingredients, materials, compositions, dosage forms, etc. that are suitable for use in contact with human tissue within the scope of sound medical judgment. Each carrier, diluent, excipient, etc. must be “acceptable” in the sense of being compatible with the other ingredients of the formulation.

  The formulation can be prepared by any method well known in the pharmaceutical arts. Such methods include the step of bringing the compound into association with a carrier which constitutes one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately bringing into association the above compounds with carriers (e.g., liquid carriers, finely divided solid carriers, etc.) and then, if necessary, shaping the product.

  Formulations can be prepared to provide immediate or sustained release; immediate, delayed, timed or sustained release; or combinations thereof.

  Formulations suitable for parenteral administration (eg, by injection) are aqueous or non-aqueous where the compound is dissolved, suspended, or otherwise provided (eg, as a liposome or other microparticle). Of an isotonic, pyrogen-free sterile solution (eg, solution, suspension). Such liquids are isotonic with antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickeners, and formulations of the recipient's blood (or other relevant body fluids). It may further include other pharmaceutically acceptable ingredients, such as solutes that render it. Examples of excipients include, for example, water, alcohol, polyol, glycerol, vegetable oil and the like. Examples of suitable isotonic carriers used in such formulations include sodium chloride injection, Ringer's solution or lactated Ringer's solution. Typically, the concentration of the compound in the liquid is from about 1 ng / ml to about 10 μg / ml, such as from about 10 ng / ml to about 1 μg / ml. The formulations can be provided in unit dose or multi-dose sealed containers, such as ampoules and vials, and are stored in a lyophilized state that requires only the addition of a sterile liquid carrier, such as water for injection, just prior to use. be able to. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

Examples of some preferred formulations One aspect of the present invention is a specification (eg, obtained or obtainable by a method described herein; having a purity described herein; etc.) Relates to a dosage unit (for example a pharmaceutical tablet or capsule) comprising 20-300 mg of a compound as described in the document and a pharmaceutically acceptable carrier, diluent or excipient.

In some embodiments, the dosage unit is a tablet.
In some embodiments, the dosage unit is a capsule.

In some embodiments, the capsule is a gelatin capsule.
In some embodiments, the capsule is an HPMC (hydroxypropyl methylcellulose) capsule.

In some embodiments, the amount is 30-200 mg.
In some embodiments, the amount is about 30 mg.
In some embodiments, the amount is about 60 mg.
In some embodiments, the amount is about 100 mg.
In some embodiments, the amount is about 150 mg.
In some embodiments, the amount is about 200 mg.

  Throughout this specification, a dose, such as those indicated above, can mean the amount of the compound itself or the free base equivalent contained in the dosage unit (ie, the amount of LMT moiety). Both of these options are clearly disclosed by the present invention.

  In some embodiments, pharmaceutically acceptable carriers, diluents, or excipients include glycerides (eg, Gelucire 44 / 14®; Lauroyl Macrogol-32 glycerides PhEur, USP) and colloidal dioxide. One or both of silicon (eg, 2% Aerosil 200®; Colloidal Silicon Dioxide PhEur, USP) or include.

New formulations— Methods commonly used for formulation and film coating of solid dosage form tablets often require the use of heat with low humidity during the drying process.

  LMTM and other leuco-methylthionium salts are potentially prone to oxidation to the methylthioninium moiety (MT), for example, decomposition to L Azure B (LAB) (see scheme below) :

  Thus, for materials such as LMTM, which tend to oxidize (as explained above), conventional formulation methods can lead to degradation and thus instability in product performance.

  Thus, the principle of the formulation of the present invention is that a leuco-methylthionium salt, such as bis (methanesulfonate) (LMTM), as an active substance, is compressed in which the active substance is present in a substantially stable form. Of pharmaceutical preparations and capsules prepared by direct tablet compression techniques or other unique tableting techniques and by encapsulation.

  The most commonly used method for the preparation of solid dosage forms is wet granulation (also called moist granulation). This includes the addition of granulating fluid to the powder. The granulation liquid may be water or some other sufficiently volatile solvent that can be removed later by drying. The granulation liquid may also contain a binder. Once the solvent is removed, the resulting mass is crushed.

  Wet granulation methods are often preferred over direct compression methods because they are more likely to overcome any problems associated with the physical properties of the various ingredients in the formulation. The wet granulation method provides a material having the fluidity and cohesiveness necessary to obtain an acceptable solid dosage form. Since all granules usually contain the same amount of drug, the content uniformity of the solid dosage form is generally improved by wet granulation. Separation of the drug from the excipient is also avoided.

  In the direct compression method, the individual components of the composition to be compressed are mixed without prior granulation and then directly compressed. While this appears to be a sophisticated and simple method, it is difficult to obtain commercially useful tablets that have sufficient strength yet disintegrate quickly enough after administration. Also, many active substances cannot be processed directly by tableting because they cannot be compressed without a granulation step.

  Surprisingly, the compounds of the present invention are stable in dry compressed solid dosage forms such as tablets during manufacture and storage, formations such as L Azure B (LAB) and methylthioninium (MT). It has now been found that the amount of degradation product made can be controlled within specifications (eg, less than 2% LAB and less than 12% MT).

  This is in contrast to, for example, the behavior of LMTM when processed by conventional wet granulation. Without wishing to be bound by theory, in conventional wet granulation processes, for example, LMTM can be very unstable and significant amounts of LAB and MT can be formed.

  Accordingly, one aspect of the invention provides a solid dosage form pharmaceutical composition comprising a compound of the invention. Preferably, the composition further comprises at least one diluent suitable for dry compression. The pharmaceutical composition is characterized in that the compound is present in a substantially stable form.

  Another aspect of the present invention is a free-flowing, agglomerated powder comprising a compound of the present invention and at least one diluent suitable for direct compression, and optionally one or more other excipients. Provided, the powder can be compressed into a solid dosage form.

  These compositions and formulations are first described herein with respect to the bis (sulfonate) salts of the invention, in particular LMTM. However, the benefits of this formulation method apply equally to other members of the leuco-methylthionium family of salts.

  For example, the formulations described herein can be used in the 3,7-diamino-10H-phenothiazinium salt disclosed in PCT International Patent Application Publication No. WO 2007/110627 (WisTa Laboratories Ltd) briefly discussed above. Is also applicable. These include leuco-methylthionium bis (hydrobromide) (LMT.2HBr, LMTB) and leuco-methylthionium bis (hydrochloride) (LMT.2HCl, LMTC).

  Accordingly, in a broader aspect, the present invention provides the following formula I:

{Where
R ¹ , R ⁹ , R ^3NA , R ^3NB , R ^7NA and R ^7NB are as defined above;
Further, where HX ¹ and HX ² are each independently a protonic acid}
Or a pharmaceutically acceptable salt, solvate or hydrate thereof, provided herein is a solid dosage form pharmaceutical composition.

  For completeness, as will be appreciated by those skilled in the art, the above equation is equally:

{Where
X ¹ and X ² are the corresponding counter ions}
Note that it can also be written as

Preferably, X ¹ and X ² are independently sulfonates or halides (Cl ⁻ , Br ⁻ , such as alkyl sulfonates or aryl sulfonates such as R ^A SO ₃ ^— or R ^B SO ₃ ^— as defined above). , I ⁻ ). In other words, HX ¹ and HX ² are preferably independently sulfonic acid (R ^A SO ₃ H, R ^B SO ₃ H) or hydrohalide (HCl, HBr, HI).

  As used hereinafter, the term “active ingredient” means the related leuco (methylthioninium) salt. In other words, this means a compound of the invention, for example a compound of formula (I) such as LMTM.

  Another aspect of the present invention provides a method for producing the above pharmaceutical composition by a dry compression method. The method preferably comprises dry compression of a dense powder mixture of the active compound and at least one diluent suitable for dry compression, and optionally one or more other excipients.

In some embodiments, the method includes direct tableting.
In some embodiments, the method includes simple direct compression.
In some embodiments, the method includes dry granulation.
In some embodiments, the method includes the moist granulation of excipients followed by the addition of the extra-granularly active ingredient.

  The solid dosage forms of the present invention advantageously exhibit the long-term chemical and physical stability of the active ingredient (a compound of the present invention, such as LMTM). The pharmaceutical composition of the present invention also has a fast dissolution rate even after prolonged storage.

  In this context, a “substantially stable” form of the active ingredient is an impurity such as an oxidative impurity or other degradation product that reacts to any significant degree during the formulation process or during storage of the formulated product. The form which does not form.

  Thus, in this context, reference may be made to materials that contain, for example, less than 20% w / w, less than 15% w / w, less than 10% w / w oxidative impurities or other degradation products. In other words, the material comprises at least 80% w / w, at least 85% w / w, at least 90% w / w of pure active ingredient in its original (unreacted) form.

  In some embodiments, the material comprising the active ingredient may comprise, for example, less than 20% w / w, less than 15% w / w, less than 12% w / w, or less than 10% w / w MT. In some embodiments, the material can include, for example, less than 5% w / w, less than 3% w / w, or less than 2% w / w LAB.

  A “stable” tablet is a tablet that, in the context of the present invention, remains substantially stable after prolonged storage under temperature and humidity controlled conditions. Stability testing can be performed with solid dosage forms that have been directly exposed to selected environmental conditions or included in the packaging.

Active ingredient content The amount of active ingredient in an uncoated composition is generally greater than about 10% w / w, but may be greater than 20% w / w, or greater than 30% w / w. it can. The amount of active ingredient is generally less than about 70% w / w in a tablet formulation, usually less than 60% w / w, or less than 50% w / w. Thus, the amount of active ingredient in an uncoated tablet core is typically from about 10% (or 20% or 30%) w / w to about 70% (or 60% or 50%) w / w. is there. When a coating is applied to the composition, as described below, the total weight of the composition is increased, and thus the percentage of active ingredients in the overall composition is somewhat reduced.

Diluent The active ingredient is not inherently compressible and may therefore require the addition of a suitable diluent to aid compression.

  Accordingly, a pharmaceutical composition of the invention will generally comprise at least 15% w / w, more usually at least 20%, at least 30%, at least 40% or at least 50% w / w diluent (single or Multiple).

  Diluents that can be used include one or more of calcium salts such as microcrystalline cellulose, lactose, mannitol, dicalcium phosphate, calcium sulfate and calcium carbonate, and sugars such as lactose, sucrose, dextrose and maltodextrin. Is mentioned.

  Preferred diluents are microcrystalline cellulose, lactose and mannitol. Spray dried forms of lactose and mannitol are particularly preferred forms for direct compression or dry granulation techniques of these compounds.

  Direct compression tablet diluent, such as one or more of microcrystalline cellulose, spray dried lactose, anhydrous lactose and mannitol, wherein the active ingredient described herein, for example a compound of the invention such as LMTM When formulated with it was unexpectedly found that the resulting solid dosage form is stable in the sense that the active ingredient remains chemically stable even after prolonged storage.

  Thus, the present invention is a low dose, medium dose or high dose tablet, eg, low dose, medium dose or high, which is stable, has a good dissolution profile, acceptable hardness and resistance to chipping and a short disintegration time. A method for preparing doses of LMTM tablets is provided.

Elution of the Compositions of the Invention Surprisingly, the inventors have found that the unique solid dosage forms described herein provide very fast elution rates.

  As explained hereinabove, without wishing to be bound by theory, it may be preferred that the active methylthioninium (MT) moiety be absorbed from the stomach and / or upper GI tract. It is done. Thus, a formulation with fast leuco (methylthioninium) salt decay and fast dissolution would be beneficial as it would deliver the maximum possible amount of drug to the intended absorption point.

  The fast elution rate of the solid dosage forms described herein allows them to dissolve rapidly in the stomach and / or upper GI tract, thus effectively providing the active ingredient there for rapid absorption. Means that.

  In some embodiments, the formulations of the present invention have at least 80% dissolution within 30 minutes, preferably at least 80% dissolution within 15 minutes, as assessed using standard pharmacopoeia methods. More preferably, it provides at least 80% elution within 10 minutes.

  In some embodiments, the formulations of the present invention have at least 90% dissolution within 30 minutes, preferably at least 90% dissolution within 15 minutes, as assessed using standard pharmacopoeia methods. More preferably, it provides at least 90% elution within 10 minutes.

  In some embodiments, the formulations of the invention have at least 95% dissolution within 30 minutes, preferably at least 95% dissolution within 15 minutes, as assessed using standard pharmacopoeia methods. More preferably, it provides at least 95% elution within 10 minutes.

  The dissolution rate can be measured by standard pharmacopeia methods as described in US Pharmacopoeia (USP) General Rules <711>. The current USP is USP34 (2011). For example, the dissolution rate for the preparation of the present invention can be measured using an apparatus according to USP dissolution apparatus 2 (Paddle).

In some embodiments, the elution rate is evaluated in LMT at a working concentration of about 5 μg / ml in 0.1 M hydrochloric acid with stirring at a paddle speed of 50 rpm. In some embodiments, the elution rate is assessed by spectrophotometric analysis. In some embodiments, the analysis includes UV / visible spectrophotometry (λ _{max LMT} = 255 nm).

  As a result of their surprisingly high dissolution rate, the formulation methods described herein can provide active compounds with a high degree of bioavailability.

  Even when storage is under “stress” conditions (ie, high temperature and humidity), a fast dissolution rate is maintained after prolonged storage. The fast dissolution rate and thus good bioavailability of the composition formulated by the method of the present invention is also highly resistant to the diversity of the formulation itself.

Other Ingredients The pharmaceutical composition will generally also include a lubricant. Examples of lubricants include magnesium stearate, calcium stearate, sodium stearyl fumarate, stearic acid, glyceryl behaptate, polyethylene glycol, ethylene oxide polymers (eg Union Carbide, Inc under the registered trademark Carbowax). , Available from Danbury, CT), sodium lauryl sulfate, magnesium lauryl stearate, a mixture of magnesium stearate and sodium lauryl sulfate, and hydrogenated vegetable oils. Preferred lubricants include calcium stearate, magnesium stearate and sodium stearyl fumarate. Most preferred as a lubricant is magnesium stearate. Lubricants generally constitute about 0.5 to about 5.0% of the total (uncoated) tablet weight. The amount of lubricant used is generally about 1.0 to about 2.0%, preferably 0.5 to 2.0% w / w.

  In addition to the diluent (s) and lubricant (s), other conventional excipients may be present in the pharmaceutical compositions of the present invention. Such additional excipients include disintegrants, binders, flavors, colorants and glidants. Some excipients can serve multiple functions, such as both as a binder and a tablet disintegrant.

  The tablet disintegrant can be present in an amount necessary to achieve rapid dissolution. Disintegrants counter the physical forces of particle binding in tablets or capsules when the dosage form is placed in an aqueous environment. Examples of disintegrants include cross-linked polyvinyl pyrrolidone (crospovidone), sodium starch glycolate, cross-linked sodium carboxymethylcellulose (croscarmellose sodium), and pregelatinized starch. Generally, the amount of disintegrant is from 0 to about 25% w / w of the composition, more usually from about 1% to about 15% w / w, and usually less than 10% or 5% w / w Is less than.

  A binder is an excipient that contributes to the adhesion of particles in a solid formulation. Examples of binders include cellulose derivatives (carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, ethylcellulose, microcrystalline cellulose) and sugars such as lactose, sucrose, dextrose, glucose, maltodextrin, and mannitol, Examples include xylitol, polymethacrylate, polyvinylpyrrolidone, sorbitol, pregelatinized starch, alginic acid and its salts such as sodium alginate, aluminum magnesium silicate, polyethylene glycol, carrageenan and the like. In general, the amount of binder can vary widely, such as from 0% to 95% w / w of the composition. As described above, excipients serve multiple functions. For example, a tableting diluent also serves as a binder.

  A glidant is a substance added to a powder to improve its flowability. Examples of glidants include magnesium stearate, colloidal silicon dioxide (such as the grade sold as Aerosil), starch and talc. The glidant can be present in the pharmaceutical composition at a level from 0 to about 5% w / w. Again, however, it should be noted that the excipient may serve multiple functions. For example, a lubricant such as magnesium stearate can also function as a glidant.

  Examples of colorants that can be incorporated into the pharmaceutical composition of the present invention include food suitable dyes such as those known as titanium dioxide and / or FD & C dyes and natural colorants. The colorant is unlikely to be used in the powder mixture compressed according to the second aspect of the invention, but can form part of a coating applied to the composition, as described below, In that case, the colorant may be present in the film coating in an amount up to about 2.0% w / w.

  Tablets are preferably coated with a conventional film coating, which provides the final product with robustness, ease of swallowing and an elegant appearance. Many polymer film coating materials are known in the art. A preferred film coating material is hydroxypropyl methylcellulose (HPMC) or partially hydrolyzed polyvinyl alcohol (PVA) (polyvinyl alcohol-part hydrated). HPMC and PVA are commercially available from Colorcon, etc., in registered Opadry coating formulations containing excipients that act as coating aids. The Opadry formulation may include talc, polydextrose, triacetin, polyethylene glycol, polysorbate 80, titanium dioxide and one or more dyes or lakes. Other suitable film-forming polymers may also be used, including vinyl copolymers such as hydroxypropylcellulose, polyvinylpyrrolidone and polyvinyl acetate and acrylic acid-methacrylic acid copolymers. The use of a film coating is beneficial for ease of handling because the blue uncoated core may stain the inside of the mouth while swallowing. The coating also improves the light stability of the dosage form.

  Tablet coating can be conveniently performed using conventional coating pans. In a preferred embodiment of the method, the coating pan is preheated with heated inflow air until the exhaust temperature reaches 35-55 ° C, more preferably 40-50 ° C. This typically may require 10-15 minutes of application of heated inlet air at an inlet temperature of 45-75 ° C, preferably 50-65 ° C. Thereafter, a tablet core containing the active ingredient (eg, LMTM) is added to the coating pan and an aqueous film coat is applied. The spray rate is controlled so that the bed temperature is maintained at 38-48 ° C, more preferably 42-44 ° C, until the desired weight gain (coating weight) is achieved.

Dry Compression Method As used herein, “dry compression” refers to a compression technique that does not involve the use of heat or moisture. Dry compression includes direct compression of the active ingredient with a suitable diluent, or it may include dry granulation (eg, slagging / double compression method or roller compression).

  Direct compression may include simple direct compression of the active ingredient and a diluent suitable for direct compression. Alternatively, excipient granulation to produce a dry granular excipient mixture that can then be directly tableted with the dry active ingredient (and optionally further dry excipients), such as Wet granulation can also be included.

  Thus, in some embodiments, the solid dosage forms of the present invention can be produced in a manufacturing process that includes simple direct compression. In this embodiment, the tablet ingredients, i.e. the active ingredient (e.g. LMTM), diluent (s) and any other excipients are mixed together in solid particulate form, such as in a tumbling blender, A dense mixture is created and then compressed using a tablet press.

  In other embodiments, the composition is prepared by dry granulation. Dry granulation means a granulation method that does not use a granulating liquid. In order for a material to be dry granulated, at least one of its components, either the active ingredient or the diluent, must have agglomeration properties. Dry granulation can be performed by a method known as “slagging”. In “slagging”, the material to be granulated is initially a tablet press (typically a linear press in US Pat. No. 4,880,373), typically equipped with a large flat-faced tooling. Is used to make a large compressed mass or “slug”. By allowing sufficient time for escape of air from the material being compressed, a fairly dense slug can be formed. The compressed slug is then crushed manually or automatically through the desired mesh screen, such as by a comminuting mill. The formation of granules by “slugging” is also known as pre-compression. If the tablet is made from granulated slugged material, this method is called a “double compression method”.

  Dry granulation can also be carried out using a “roller compressor”. In a roller compressor, material particles are compacted and compressed by passing between two high pressure rollers. The compressed material from the roller compressor is then reduced to a uniform particle size by grinding. The uniform granules can then be mixed with other substances such as lubricants to compress the material (eg, by a rotary tablet press). In addition to pharmaceutical applications, roller compaction is used in other industries such as the food industry, animal feed industry and fertilizer industry.

Today, dry granulation is generally understood to mean roller compaction or slagging and is well known to those skilled in the art (eg, Pharmaceutical Dosage Forms: Tablets (Leiberman, Lachman, and Schwartz (Eds); Marcel Dekker, Inc, ^{2 nd Edition, 1989) andRemington's} Pharmaceutical Sciences (AR Gennaro (Ed); Mack Publishing Co, Easton, PA 18 th edition, 1990) see).

  In a further embodiment of the invention, the tablets are prepared by wet granulation of excipients and incorporation of MTC while further granulating. Typically, such methods include lactose and / or microcrystalline cellulose optionally with a binder such as polyvinylpyrrolidone and a wet massing diluent such as water. The wet mass is dried and then passed through a mesh to form granules. Any remaining excipients such as the active ingredient and lubricant are then mixed with the dried granules and compressed to form tablets.

Use of Acids in Compositions of the Invention Compositions comprising leuco (methylthioninium) compounds, including compounds of the invention such as LMTM, in some embodiments, are suitable in certain amounts of certain amounts prior to formulation. It can be stabilized by adding acid to the bulk substance. In order to provide a stable pharmaceutical composition for the purpose of preventing further MT formation throughout the formulation and throughout the product's lifetime, thereby obtaining regulatory approval and reducing the associated costs in packaging. Can be used.

  Thus, according to the present invention, a pharmaceutical composition comprising an active ingredient as described herein and a pharmaceutically acceptable carrier, the formulation further comprising a sufficient amount of acid to prevent MT formation A pharmaceutical composition is also provided. Without wishing to be bound by theory, an acid having a pK1 greater than 1.5 is considered preferred. In some embodiments, the acid is present in an amount from 5% to 25% w / w.

  Preferably, the composition is prepared by the dry compression method described above.

  Preferred acids for the purposes of the present invention are maleic acid (pK1 1.9), phosphoric acid (pK1 2.12), ascorbic acid (pK1 4.17), sorbic acid (pK1 4.76), aspartic acid and Sialic acid. The stabilizing action of the added acid can be enhanced by choosing an appropriate carrier. The carrier is preferably mannitol, cellulosic material, or starch, or a mixture thereof. Typically, the carrier is present in an amount of at least 40% w / w of the formulation.

By choosing an appropriate particle size range for the dry powder mixture, typically more than 10% of the particles have a size greater than 10 microns, a significant reduction in MT formation can also be achieved. It was found. Thus, according to another aspect of the invention, a pharmaceutical composition comprising an active ingredient as described herein and a pharmaceutically acceptable carrier, the composition comprising more than 10% greater than 10 microns. There is provided a pharmaceutical composition further comprising particles having a size.

Carriers It has been found that significant reductions in MT formation can also be achieved by selection of appropriate carriers, particularly those having a particle shape that resists water penetration. For example, Elcema ™, a smooth, flat, long lamellar particle with a nonporous surface, appears to reduce MT formation by restricting water access. For this purpose, ethylcellulose, mannitol and Starch 1500 ™ and microcrystalline cellulose are also particularly suitable.

  Thus, according to another aspect of the present invention, there is provided a pharmaceutical composition comprising a leuco (methylthioninium) compound, for example a compound of the present invention such as LMTM, and a pharmaceutically acceptable carrier, wherein the carrier is Elcema. A pharmaceutical composition is provided, characterized in that it is TM, ethylcellulose, mannitol, or Starch 1500 TM.

Encapsulation The stabilized dry powder mixture according to the present invention is compressed into tablets, for example (either previously converted to granulated powder, etc. by means such as described in Formulation Examples 1-4, or not) Or filled into capsules, resulting in a pharmaceutical composition having a very good shelf life.

  The capsules according to the invention are typically made of gelatin, preferably HPMC. Preferred excipients include lactose, starch, cellulose, lactose and high molecular weight polyethylene glycols.

CONCLUSION Pharmaceutical compositions and formulations prepared by the above method are more stable immediately after completion of manufacture than formulations produced using conventional aqueous granulation methods. Furthermore, they can show enhanced stability in storage.

  For example, a pharmaceutical composition comprising LMTM with a content of 10-50% by weight, preferably 15% -40% by weight, prepared in this way can be subjected to standard stability tests, eg long-term accelerated stability. In the test, at a temperature of 25 ° C. and a relative humidity of 60 ± 5%, the increase in L Azure B within 24 months does not exceed 2% relative to the LMTM peak area.

  During processing and storage, leuco (methylthioninium) compounds such as LMTM can oxidize to produce small amounts of MT (see scheme above).

  Relatively low concentrations (eg, less than 12%) of MT in the leuco formulations of the present invention are not desirable, but were provided to the body as charged or oxidized forms from LMTM and various other leuco salts. Even so, it cannot be considered to have detrimental clinical significance by itself because it can then be reduced to the uncharged (reduced) MT form before being absorbed. In addition to the small amount of MT formed during processing such as mixing and tableting, the leuco-methylthioninium salt of the present invention is absorbed by excipients present in the tablet, especially in the presence of moisture. React with oxygen to produce more MT.

  One benefit of the formulations of the present invention is to minimize the amount of MT formed in the tablet to less than 12% over 2 years when stored at, for example, 25 ° C. and 60% relative humidity. It is. This means the accumulated amount of MT formed during both tablet processing and storage: In general, the formulation method of the present invention results in less than 5% MT formation during processing; , Meaning that up to about 5-7% MT is formed during storage of the finished pack. This provides a shelf life of at least 24 months.

  This is demonstrated in the following formulation examples.

It will be appreciated by those skilled in the art that the appropriate dosage of the above compounds and compositions containing such compounds can vary from patient to patient. The determination of the optimal dose will generally involve a balance of therapeutic effects for any risk or adverse side effects. The selected dose level will depend on the activity of the particular compound, route of administration, administration time, excretion rate of the compound, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and patient species, gender Will depend on a variety of factors, including but not limited to, age, weight, condition, general health, and prior medical history. In general, the dosage will be selected to achieve a local concentration at the site of action that achieves the desired effect without substantially harming or deleterious side effects, but the amount and administration of the compound The route will ultimately be at the discretion of the physician, veterinarian or clinician.

  Administration can occur continuously throughout the course of treatment, in a single dose, or intermittently (eg, in divided doses at appropriate intervals). Methods of determining the most effective means and dose of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian or clinician.

  In general, a suitable dose of the compound is about 100 ng to 25 mg (more typically about 1 μg to about 10 mg) per kilogram of subject body weight per day.

  In some embodiments, the compound is administered to a human patient according to the following dosage regime: about 100 mg, 3 times daily.

  In some embodiments, the compound is administered to a human patient according to the following dosage regime: about 150 mg, twice daily.

  In some embodiments, the compound is administered to a human patient according to the following dosage regime: about 200 mg, twice daily.

  The following examples are provided only to illustrate the present invention and are not intended to limit the scope thereof.

Example 1 Synthesis and Characterization
Laboratory synthesis of 10-acetyl-N, N, N ', N'-tetramethylphenothiazine-3,7-diamine

Synthesis of 3,7-dinitro-10H-phenothiazine (2)
To a 1 liter three-necked round bottom flask (RBF) equipped with a thermometer, dropping funnel and condenser, was added phenothiazine (molecular weight 199, 28 g / mol, 25.00 g, 125.5 mmol) and dimethyl sulfoxide (250 ml). Add and stir the mixture for 2 minutes or until the phenothiazine is dissolved. The condenser was then connected to a Dreschel bottle half-filled with water. Sodium nitrite (molecular weight 69.00 g / mol, 51.94 g, 752.7 mmol) was added to RBF and acetic acid (150 ml) was added to the dropping funnel. Acetic acid was then added dropwise to the RBF over 20 minutes. The light yellow slurry turned red and solids precipitated from the solution. After the acetic acid addition was complete, the mixture was stirred at ambient temperature (36-20 ° C.) for 2 hours, after which the temperature was raised to 95 ° C. and stirred for 17 hours. After this time, the mixture was cooled to 50 ° C., methanol (100 ml) was added and the mixture was further cooled to 22 ° C. The cooled mixture was then filtered and the cake was washed with methanol (3 × 25 ml). The washed cake with vacuum applied was left on the filter for 30 minutes and then dried at 50 ° C. for 15 hours to yield the product as a brown solid. (Molecular weight 289.27 g / mol, 29.45 g, 81%).

Remarks 1. The addition of acetic acid yielded NOx gas, which was converted to nitric acid by bubbling this gas in a dresser bottle half full of water.
2. The addition of acetic acid was exothermic and the mixture rose from 22 ° C to 36 ° C.
3. Methanol was added to help dissolve any sodium acetate and as an anti-solvent to maximize product yield.
4). The synthesis was successful even when dimethylformamide (DMF), acetonitrile (MeCN), tetrahydrofuran (THF), acetone or dimethoxyethane (DME) was used as the reaction solvent.

  NMR: The product (5 mg) is dissolved in DMSO-d6 (1.5 ml) and may need to be warmed to completely dissolve the solid.

Synthesis of 3,7-dinitro-10-acetylphenothiazine (3)
To a 500 ml three-necked round bottom flask equipped with a thermometer and a condenser, 3,7-dinitro-10H-phenothiazine (molecular weight 289.27 g / mol, 29.00 g, 100 mmol), dimethylformamide (58 ml), anhydrous Acetic acid (molecular weight 102.09 g / mol, 102.09 g, 1000 mmol) and triethylamine (molecular weight 101.19 g / mol, 40.88 g, 401 mmol) were added. The mixture was heated to 105 ° C. and stirred at this temperature for 3 hours. The mixture was cooled to ambient temperature (21 ° C.) and then stirred for 1 hour until cooled to 5 ° C. The product was isolated by filtration and washed with methanol (3 × 30 ml) to give a bright yellow crystalline solid which was dried at 50 ° C. for 15 hours (molecular weight 331.31 g / mol, 26.94 g, 81 %).

Remarks 1. Product crystals form after about 1 hour at 105 ° C. during the reaction.
2. After cooling, the product bulk precipitates at about 70 ° C.
3. The product was orange before washing with methanol.

  NMR: The product (10 mg) was dissolved in DMSO-d6 (1.5 ml).

Synthesis of 10-acetyl-N, N, N ′, N′-tetramethylphenothiazine-3,7-diamine (5)
A 100 ml three-necked round bottom flask equipped with a thermometer and condenser was charged with 3,7-dinitro-10-acetylphenothiazine (molecular weight 331.31 g / mol, 5 g, 15.09 mmol), palladium on carbon (10%, Dry, 0.5 g) and 2-methyltetrahydrofuran (25 ml) were added. The flask was evacuated and purged with hydrogen five times before heating the mixture to 56 ° C. After 17 hours, it was determined that the reduction was complete to yield compound 4 (see tlc conditions) and formalin was added (molecular weight 30.03 g / mol, 14.7 g, 181.1 mmol). The flask was evacuated once more and purged with hydrogen 5 times. 71 hours after the addition of formalin (total time 88 hours), the completion of tetra-methylation was determined by tlc at 56 ° C. The mixture was filtered at 50 ° C., the gray catalyst was washed with 2-methyltetrahydrofuran (3 × 5 ml), and the filtrate and washings were combined. Methanol (5 ml) was added to this solution to homogenize the mixture. Upon cooling to 5 ° C., a colorless solid precipitated from the solution. Two more volumes of methanol (10 ml) were added and the slurry was stirred at 5 ° C. for 50 minutes. The crude product was isolated by filtration to give a colorless solid, which was washed with methanol (3 × 5 ml) and dried at 50 ° C. for 16 hours (molecular weight 327.45 g / mol, 2.26 g, 46% ). Addition of water (50 ml) to the filtrate from the isolation process resulted in more solids. The suspension was stirred at 5 ° C. for 2 hours, then collected by filtration, washed with methanol (3 × 5 ml) and dried at 50 ° C. for 13 hours (molecular weight 327.45 g / mol, 0.83 g, 17%). The total yield of product was (3.09 g, 63%).

Remarks 1. Normal phase tlc conditions, eluent 75% ethyl acetate, 25% petroleum refinery (40-60 ° C), and 254 nm UV lamp.
2. The retention factor for the dinitro starting material is 0.68 as a yellow spot, the retention factor for the hydrogenated product is 0.25 as the blue spot, and the retention factor for the methylated product is light blue. The spot is 0.67.
3. The method for tlc analysis of the hydrogenation step was direct spotting, but analysis of the methylated product included water added to the reaction aliquot, which was extracted and spotted with ethyl acetate.
After 4.17 hours, tlc shows two spots, the main spot is the reduction product and the minor spot is unknown.
After 5.88 hours, tlc showed mainly tetra-methylated product as the main spot.
6). Typically, the reduction and methylation will be complete within 72 hours.
7. 1H NMR spectroscopy of the two samples produced identical spectra and detected traces of 2-methyltetrahydrofuran with an unknown signal at 5 ppm.

NMR: The product (10 mg) was dissolved in CDCl ₃ (1.5 ml).

Alternative synthesis of 10-acetyl-N, N, N ′, N′-tetramethylphenothiazine-3,7-diamine (5)
A 50 ml round bottom flask was charged with 3,7-dinitro-10H-acetylphenothiazine (molecular weight 331.31 g / mol, 1 g, 3.02 mmol), zinc dust (molecular weight 65.39 g / mol, 1.38 g, 21.13). Mmol), methanol (6 ml) and tetrahydrofuran (2 ml) were added. The mixture was heated to 50 ° C., then a warm solution (45-50 ° C.) of aqueous ammonium chloride (molecular weight 53.49 g / mol, 2.26 g, 42.26 mmol dissolved in 6 ml of water) was added slowly. To maintain a gentle reflux. The mixture was then heated to 70 ° C. and stirred at this temperature for 2 hours before cooling to ambient temperature (23 ° C.). The cooled mixture was filtered to remove the zinc salt and the filtrate containing compound 4 was paraformaldehyde (molecular weight 30.03 g / mol, 1.09 g, 36.22 mmol), sodium cyanoborohydride (molecular weight 62.84 g). / Mol, 1.14 g, 18.11 mmol) and acetic acid (2 ml). The mixture was heated to 50 ° C. and stirred at this temperature for 3 hours. After cooling to ambient temperature (23 ° C.), water (2 × 10 ml) was added and the colorless slurry was stirred for 16 hours. The solid was then collected by filtration and washed with methanol (3 × 2 ml) to give the title compound (molecular weight 327.45 g / mol, 0.91 g, 92%) as an off-white solid.

Remarks 1. The reduction reaction using zinc and aqueous ammonium chloride was fast and clean, taking only 2 hours to complete, and no other spots were recorded by tlc analysis.
2. Reductive methylation with sodium cyanoborohydride, paraformaldehyde and acetic acid was fast and clean and took only 3 hours to complete.

NMR: The product (10 mg) was dissolved in CDCl ₃ (1.5 ml).

Synthesis 1: Synthesis of N, N, N ', N'-tetramethyl-10H-phenothiazine-3,7-diaminium bis (methanesulfonate) (LMT.2MsOH)

10-acetyl-N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diamine (AcMT) (150 g) was added to a three-necked round bottom flask. Toluene (1.8 l) was added and the mixture was heated to reflux for 30 minutes. The solution was allowed to cool to 70 ° C. before passing through a 5 μ in-line filter into a jacketed vessel equipped with distillation apparatus ¹ . Toluene (150 ml) was added to the round bottom flask. This was used to rinse the transfer line and filter. Under reduced pressure ² , about 1.4 l of toluene was distilled off. The temperature was lowered to 18 ° C. ³ before adding water (42 ml). This was followed by the addition of ⁴ methanesulfonic acid (MSA) (65.5 ml, 99%. 2.2 eq) over 5 minutes. A second portion of water (18 ml) was added. The mixture was heated to 85 ° C. for 3 hours by which time the reaction was judged complete by tlc analysis. The biphasic solution was allowed to cool to 50 ° C. ⁵ before anhydrous EtOH (150 ml) was added over 20 minutes. 150 mg of N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diaminium bis (methanesulfonate) was seeded into the mixture ^6-8 . A second portion of EtOH (600 ml) was added over 90 minutes ⁹ and the reaction was allowed to cool to 20 ° C. over 1 hour ¹⁰ . It was stirred at this temperature for 1 hour before the solid was collected by filtration. The cake was washed with 3 × 300 ml of MeCN ¹¹ , aspirated for 5 minutes until dry and placed under vacuum overnight to give the product as a yellow crystalline solid (yield 85-90%).

Remarks 1. Heating to reflux temperature ensures complete dissolution of AcMT for transfer through the 5μ filter. Toluene is a good solvent and targeting 70 ° C. ensures that the material stays in solution and minimizes the possibility of damage to the plastic transfer hose and filter Is a compromise between the two.
2. 500 ml of remaining toluene ensures that the reaction volume meets the minimum agitation depth of the reaction vessel.
3. The volume of water was controlled to ensure that the product crystallized as a free-flowing precipitate. Reaction seeding reduces the effect of small fluctuations in water volume.
4). To carry out the hydrolysis and form a salt leaving a sufficient amount of excess acid (0.2 eq) to ensure the stability of the product in solution, 2.2 eq of MSA was added. use. The addition of MSA causes a slight exotherm and therefore the addition time is 5 minutes.
5. EtOH is used as a counter solvent for precipitating the product. To ensure that the seed does not dissolve, a portion is added before the seed. Extension of the addition time ensures controlled crystallization of the product (see notes 7 and 8).
6). It is possible to carry out the reaction without the use of seeds, but its uptake is LMT. Ensuring premature precipitation of 2MsOH, which in turn is a potential genotoxic byproduct alcohol ester EMS—inclusion of byproducts and EtOH inclusion (not detected in the synthesis process, etc.) To prevent.
7). Seeds are also useful as a means of controlling product particle size. When seed material ground to less than 100 μm with a mortar and pestle is used, a significant reduction in the average particle size of the product is observed. No such effect was observed when using less than 100 μm seed that was not ground. Thus, without wishing to be bound by theory, the ability of the seed to control the grain size is not a function of the seed grain size, but the proportion of the “new” crystal plane exposed by the original or seed fracture. It seems to be linked.
8). Finally, if the seed material is relatively large and not crushed, a significant amount of product (thin film) can adhere to the sides of the reaction vessel during EtOH addition. This can be reduced by introducing a heating / cooling cycle into the process after the EtOH addition. However, an unexpected bonus to the use of crushed seed was to reduce the level of thinned material present after EtOH addition by about 90%. It was therefore no longer necessary to carry out a heating / cooling cycle. This appears to be linked to a smaller seed size than the new surface, since a similar reduction in coating was observed when the reaction was performed with unbroken seeds less than 100μ. .
9. The rate of EtOH addition has an effect on particle size and inclusion of EtOH. Rapid addition (less than 1 hour) reduces particle size but increases EtOH encapsulation. Slow addition (2 hours) has the opposite effect and therefore must be balanced.
10. The cooling rate is similar but has a lower effect. Rapid cooling (less than 1 hour) results in a decrease in particle size and a concomitant increase in EtOH levels. Slow cooling has the opposite effect.
11. EtOH is equally effective as MeCN in removing related substances, but its use is accompanied by a slight increase in the level of retained EtOH.

Characterization elemental analysis of N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diaminium bis (methanesulfonate) (LMT.2MsOH) (microanalysis)
The analysis has a good correlation between the theoretical and analytical values for carbon, nitrogen, hydrogen and sulfur.

¹ H-nuclear magnetic resonance (NMR) spectroscopy
¹ H-NMR spectra were obtained on a Varian 600 MHz instrument in deuterated methanol CD ₃ OD and are shown in FIG.

The assignment of the ¹ H NMR spectrum is as follows:

¹³ C-nuclear magnetic resonance (NMR) spectroscopy
¹³ C NMR spectra were obtained on a Varian 400 MHz NMR instrument in deuterated methanol CD ₃ OD at a frequency of 100.56 MHz and are shown in FIG.

The first assignment of the ¹³ C-NMR spectrum was based on a correlation with a chart of known chemical shifts (reference: Structure Determination of Organic Compounds: Tables of Spectral Data, Pretsch E., et al. Springer, London, p122). .

  Further assignment utilized DEPT-135, HSQC and HMBC experiments to clearly confirm the assignment. DEPT-135 (Distortionless Enhancement by Polarization Transfer), HSQC (Heteronuclear Single Quantum Coherence) and HMBC (Heterontial Multiple Bond) )

Infrared spectroscopy (IR)
The sample was thoroughly mixed and ground with a mortar and pestle with 200 mg of anhydrous KBr. The mixture was then compressed into a disc using a die at a pressure of 1500 psi. IR spectra were then obtained on a Nicolet Avatar 320 FT-IR spectrometer. The spectrum is shown in FIG.

Mass spectrometry (MS)
Mass spectroscopic analysis was performed using a Waters, LCT Premier XE mass spectrometer. A flow rate of 1 ml / hour was employed. The source used for active ingredient analysis was positive mode electron impact ionization. The source used for analysis of methanesulfonate counterion was positive mode electrospray ionization.

Using electron impact ionization, a major peak is observed at 285 (see FIG. 7). This corresponds to the molecular ion C ₁₆ H ₁₉ N ₃ S. A comparison of the actual mass measured and the theoretical value is provided below:

The exact mass measured was in good agreement with the calculated mass for C ₁₆ H ₁₉ N ₃ S.

Using electrospray ionization, a major peak is observed at 95 (see FIG. 8). This corresponds to the molecular ion of the counter ion CH ₃ O ₃ S.

Ultraviolet-visible spectroscopy (UV-Vis)
A 5 mg sample was dissolved in deionized water to a total of 100 ml in a flask with memory. Analyzes were performed on a Perkin Elmer Lambda 25 UV / Vis spectrometer using a quartz cuvette. The UV-Vis spectrum is shown in FIG.

  The attenuation coefficient ε for λmax at 255 was 34254.64. This was calculated according to the following Lambert-Beer law:

High performance liquid chromatography (HPLC)
A 100 mg sample was submitted for HPLC analysis. Analysis was performed on an Agilent 1200 series equipped with a VWD Detector or PDA for identity according to the method summarized in the table below.

  The HPLC trace is shown in FIG. The organic substance purity was confirmed to be 99.45% w / w.

Crystal form In the above method, LMT. 2MsOH is produced in crystalline form. LMT. The crystalline form of 2MsOH is illustrated by the X-ray powder diffraction spectrum shown in FIG. XRPD shows a sharp signal that is indicative of a high degree of crystal order. A change in relative peak intensity is observed, which can be attributed to orientation effects as well as particle size differences. Only slight changes in relative peak intensity (less than 50%) are observed as a function of sample thickness (0.1 mm vs. 1.0 mm).

  The crystalline form is further characterized by FT-Raman, thermogravimetry (TG), differential scanning calorimetry (DSC), dynamic water vapor adsorption (DVS) analysis, and microscopy (FIGS. 12-16). This form can conveniently be referred to as the “A form”.

  Crystals for single crystal X-ray analysis were obtained from ethanol, methanesulfonic acid and water. See Figure 17c.

Equipment details
X-ray powder diffraction : Bruker 08 Advance, Cu Ka irradiation (λ = 1.54180 mm), 40 kV / 40 mA, LynxEye detector, 2θ step width 0.02 °, 37 seconds per step, 2θ scan range 2.5 ° to 50 °. Samples were prepared on a silicon single crystal sample holder with a depth of 0.1 or 1.0 mm without any special treatment except applying a slight pressure to achieve a flat surface. All samples were rotated during the measurement.
Differential scanning calorimetry : Perkin Elmer DSC 7. Under N _2, sealed gold crucible, heating rate 20 ° C. / min, scan to 280 ° C. from -50 ° C..
Dynamic water vapor adsorption : Projekt Messtechnik SPS 11-100n water vapor adsorption analyzer. Place the sample in an aluminum crucible on a microbalance and place a predetermined humidity program at 25 ° C. (relative humidity 50-0 to 95-50%, Δrelative humidity = 5% time ⁻¹ and “extreme” etc. Equilibration was performed at 25 ° C. and 50% relative humidity prior to initiating the “isohumid” equilibration period.
FT-Raman spectroscopy : Bruker RFS100. Nd: YAG 1064 nm excitation, 50 mW laser power, Ge detector, 128 scans, range 50~3500cm ^{-1, 2cm -1} resolution, Aluminum sample holder.
Polarization microscopy : Leitz Orthoplan microscope equipped with a Leica OFC280 CCO camera.
TG : TA Instruments TGA Q5000. Open aluminum crucible, N ₂ atmosphere, heating rate 10 ° C. min ⁻¹ , range 25-300 ° C.
TG-FTIR : Netzsch Thermo-Microbalance TG 209 equipped with a Bruker FT-IR Spectrometer Vector 22. Aluminum crucible with microholes, N ₂ atmosphere, heating rate 10 ° C. min ⁻¹ , range 25-250 ° C.

  Without wishing to be bound by theory, this form is the LMT. It is suggested that it represents the only stable polymorph of 2MsOH. Polymorphism testing shows that Form A is reproduced in almost all crystallization systems (tested in an inert atmosphere using degassed solvent).

  LMT. Amorphous LMT. 2MsOH can be prepared, but after further drying, the amorphous material recrystallizes to Form A.

AcMT and LMT. Industrial scale synthesis of 2MsOH

Large scale synthesis of 10-acetyl-N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diamine (AcMT) Acetonitrile (MeCN) (300 l) was added to reactor 1 (R1), Cooled to -5 to 0 ° C. Methylthioninium chloride trihydrate (MTC.3H ₂ O) (150 kg) was added and the temperature was raised to 15-25 ° C. Triethylamine (Et ₃ N) (100 l) was added followed by MeCN (20 l). Hydrazine hydrate (N ₂ H ₄ .H ₂ O) (12 l) was added over 30 minutes. The reaction temperature was raised to 60-70 ° C. over 1 hour and then maintained at this temperature for 1 hour before being lowered to 40-50 ° C. Acetic anhydride (Ac ₂ O) (240 l) was added over 1 h followed by a rinse with MeCN (20 l). The batch temperature was increased to 90-100 ° C. for 2 hours. The temperature was reduced to 55-65 ° C. and water (340 l) was added over 2 hours while maintaining the temperature. The batch temperature was then reduced to -5-5 ° C over 2 hours and maintained at that temperature for 6 hours. The solid was collected by filtration. The liquid was completely de-liquore from the cake before adding water (400 l) to R1. The temperature in R1 was allowed to rise to 15-25 ° C., and then water was added in portions to wash the filter cake. Prior to removal, the product was dried under a stream of nitrogen for 6 hours (yield: 90-110 kg).

Large scale purification of 10-acetyl-N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diamine (AcMT) Water (300 l) followed by 10-acetyl-N, N, N ′ , N′-Tetramethyl-10H-phenothiazine-3,7-diamine (AcMT) (100 kg) was added to R1. Toluene (400 l) and 80% aqueous acetic acid (40 l) were added followed by a water rinse (50 l). The batch temperature was raised to 75-85 ° C. for 1 hour. The stirrer was stopped and the layer was allowed to settle for 30 minutes. The lower aqueous layer was removed and fresh water (300 l), 80% aqueous acetic acid (40 l) was added, followed by rinse water (50 l). After stirring the mixture at 75-85 ° C. for 1 hour, the stirrer was stopped and the layers were allowed to settle for 30 minutes. The lower aqueous layer was removed and fresh water (300 l), 80% aqueous acetic acid (40 l) was added, followed by rinse water (50 l). After the mixture was stirred at 75-85 ° C. for 1 hour, the stirrer was stopped. After letting the layers settle for 30 minutes, the lower layer was removed, water (390 l) was added and the mixture was stirred for 1 hour. The stirrer was stopped and the layer was allowed to settle for 30 minutes. The lower aqueous layer was removed and the temperature was lowered to -5-5 ° C. The jacket temperature was raised to 80 ° C. and then when it reached 60 ° C., the temperature was reduced to −10 to 0 ° C. over 2 hours. The mixture was stirred for 4 hours and then transferred to a filter. After complete liquid removal from the cake, toluene (150 l) was added to R1. After stirring the toluene in R1 for 30 minutes, it was used separately to wash the filter cake. The product was removed after drying on the filter under nitrogen flow for less than 1% loss due to drying (yield: 75-90 kg).

Large scale synthesis of N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diaminium bis (methanesulfonate) (LMT.2MsOH) AcMT (18-22 kg) was added to R1. Toluene (volume (l) = 16 × AcMT weight) was added and the mixture was heated to 90-100 ° C. for 30 minutes. The solution was allowed to cool to 60-80 ° C. and then passed through a 5 μ in-line filter to Reactor 2 (R2). Toluene (50 l) was added to reactor 1 (about 70 ° C.) and stirred for 30 minutes. This was used to rinse the transfer line and filter. The above process was repeated once more. Next, the process of removing excess toluene from R2 by distillation under reduced pressure was started. Further, the limit capacity of R2 that allowed two doses of AcMT (18-22 kg each) was transferred from R1 to R2 according to the above method. The distillation was completed when the batch volume in R2 was reduced to about 340 l. The temperature was raised to 95-105 ° C. for 15-30 minutes and then cooled to 15-25 ° C. Water (20 l) was added to R2. This was followed by the addition of methanesulfonic acid (MSA) (331, 99%, 2.2 eq) while maintaining the batch temperature at 15-30 ° C. A second portion of water (10 l) was added and the mixture was stirred at this temperature for 2 hours. The mixture was heated to 80-90 ° C. for 3-4 hours. After the biphasic solution was allowed to cool to 48-58 ° C., absolute EtOH (75 l) was added over 15-30 minutes. The stirrer was stopped and seeded into the mixture using 150 g of crushed (less than 100 μ) N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diaminium bis (methanesulfonate). A second EtOH (300 l) was added over 80-110 minutes. The jacket temperature was set to 10 ° C and when the temperature reached 25 ° C, the jacket temperature was reset to 20 ° C. After it was stirred at 15-25 ° C. for 2 hours, the solid was collected by filtration. The liquid was completely removed from the cake. MeCN (300 l) was added to R2 and stirred for 15 minutes before being used separately to wash the filter cake. A second 300 l of MeCN was added to R2 and the washing process was repeated. The product was removed on the filter after drying loss to less than 0.2% (yield 80-90%).

Synthesis 2: Synthesis and analysis of N, N, N ', N'-tetramethyl-10H-phenothiazine-3,7-diaminium bis (ethanesulfonate) (LMT.2EsOH)

LMT. Synthetic method for 2EsOH LMT. The synthesis of 2EsOH was performed by acid hydrolysis of 10-acetyl-N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diamine. The acid used was ethanesulfonic acid and the solvent combination was aqueous methanol.

Experimental Details A 100 ml round bottom flask was charged with 10-acetyl-N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diamine (5 g, 15.27 mmol, molecular weight 327.45 g / mol). ) (70%, aqueous), ethanesulfonic acid (7.21 g, 45.81 mmol, molecular weight 110.13 g / mol) and methanol (25 ml) were added. The mixture was heated to 75 ° C. and stirred at this temperature for 4 hours, after which the mixture was cooled over ice water. A solid did not form and removal of methanol under vacuum resulted in a viscous green oil. To this oil was added isopropanol (25 ml) and the mixture was heated to reflux temperature to ensure a homogeneous solution. After cooling, acetone was added until a precipitate formed. The suspension was cooled on ice water for 1 hour and then filtered to give the crude product as a yellow solid that turned green upon exposure to air. The crude was washed with acetone (3 × 5 ml) and air dried for 3 days to yield the crude product (3.35 g, 43%, molecular weight 505.68 g / mol) as a light green solid.

Crystallography 1 g of LMT. A 2EsOH sample was dissolved in acetic acid (ca. 0.1 g) and layered with ethyl acetate and allowed to diffuse slowly in the dark for 3 days. Crystals formed and were collected and analyzed by X-ray diffraction to confirm that the product was bis (ethane sulfonate). See Figure 17a.

Synthesis 3: Synthesis and analysis of N, N, N ', N'-tetramethyl-10H-phenothiazine-3,7-diaminium bis (p-toluenesulfonate) (LMT.2TsOH)

LMT. Synthetic method for 2TsOH LMT. Synthesis of 2TsOH was performed by neutralizing N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diaminium dichloride with sodium carbonate and extracting neutral species into an organic solvent. did. The extract was treated with p-toluenesulfonic acid and the mixture was concentrated to dryness.

Experimental Details Sodium carbonate (0.59 g, 5.58 mmol, molecular weight 105.99 g / mol) and water (10 ml) were added to a 50 ml beaker and the mixture was stirred until the solid dissolved. To a 100 ml separatory funnel, N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diaminium dichloride (1 g, 2.79 mmol, molecular weight 358.33 g / mol), tetrahydrofuran (35 ml) And diethyl ether (5 ml) were added followed by an aqueous solution of sodium carbonate. Neutral species were extracted into the organic solvent layer and separated from the aqueous layer. P-Toluenesulfonic acid monohydrate (1.06 g, 5.58 mmol, molecular weight 190.20 g / mol) pre-dissolved in tetrahydrofuran (5 ml) is added to the organic extract and the mixture is concentrated to dryness. The product was produced as a crisp green amorphous foam (molecular weight 629.8216 g / mol).

Synthesis 4: Synthesis and analysis of N, N, N ', N'-tetramethyl-10H-phenothiazine-3,7-diaminium ethane disulfonate (LMT.EDSA)

  By acid hydrolysis of 10-acetyl-N, N, N ', N'-tetramethyl-10H-phenothiazine-3,7-diamine, LMT. Synthesis of EDSA was performed. The acid used was 1,2-ethanedisulfonic acid and the solvent combination was aqueous ethanol.

Experimental Details A 25 ml round bottom flask was charged with 10-acetyl-N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diamine (1 g, 3.05 mmol, molecular weight 327.45 g / mol). ), 1,2-ethanedisulfonic acid monohydrate (0.95 g, 4.58 mmol, molecular weight 208.21 g / mol), water (1 ml) and ethanol (5 ml) were added. The mixture was heated to 85 ° C. and stirred at this temperature for 2.5 hours, where a yellow-green solid precipitated from the solution. The slurry was cooled on ice water for 30 minutes and then filtered to yield the crude product as a yellow-green solid. The crude was washed with ethanol (3 × 3 ml), air dried for 15 minutes, then oven dried at a temperature of 70 ° C. for 3.5 hours to yield the crude product (1.33 g, 91%, A molecular weight of 475.61 g / mol).

LMT. Purification of EDSA In a 50 ml conical flask, crude LMT. EDSA (1 g, 2.10 mmol, molecular weight 475.61 g / mol) and water (10 ml) were added. The slurry was heated to 95 ° C. and stirred at this temperature until the solid dissolved. The solution was then allowed to cool to 25 ° C. where a bright green crystalline solid formed. The slurry was then cooled on ice water for 30 minutes and then filtered. The collected solid was washed with methanol (3 × 3 ml) and air dried for 18 hours to yield the purified product as a crystalline light green solid (0.88 g, 88%, molecular weight 475.61 g / Mole).

Crystallography 40 mg LMT. The EDSA sample was dissolved in hot deuterated water (about 1 ml) and allowed to cool slowly in the dark. The generated crystals were collected and analyzed by X-ray diffraction to confirm that the product was LMT vs. EDSA adduct 1: 1 monohydrate. See Figure 17b.

Synthesis 5: Synthesis and analysis of N, N, N ', N'-tetramethyl-10H-phenothiazine-3,7-diaminium naphthalene disulfonate (LMT.NDSA)

LMT. Synthesis Method for NDSA LMT. The synthesis of NDSA was performed by acid hydrolysis of 10-acetyl-N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diamine. The acid used was 1,5-naphthalenedisulfonic acid and the solvent combination was aqueous ethanol.

Experimental Details In a 25 ml round bottom flask was added 10-acetyl-N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diamine (1 g, 3.05 mmol, molecular weight 327.45 g / mol). ), 1,5-naphthalenedisulfonic acid tetrahydrate (1.65 g, 4.58 mmol, molecular weight 3600.36 g / mol), water (1 ml) and ethanol (5 ml) were added. The mixture was heated to 85 ° C. and stirred at this temperature for 30 minutes, where the mixture was still insoluble. To the hot mixture was added water (4 ml) and the reaction was heated to 95 ° C. and stirred at this temperature for 8 hours. The suspension was cooled on ice water for 10 minutes and then filtered to yield the crude product as a light green solid. The crude was washed with ethanol (3 × 5 ml) and air dried for 3 days to yield the crude product (1.75 g, 100%, molecular weight 573.71 g / mol) as a light blue-green solid.

Example 2-Solubility Test i) N, N, N ', N'-Tetramethyl-10H-phenothiazine-3,7-diaminium dibromide, dichloride and bis (methanesulfonate) (LMT.2HBr, LMT.2HCl and LMT.2MsOH) Salt Solubility Two aqueous solutions (at 21.4 ° C., pH 2.00 and 3.01) were prepared by carefully adding HCl (5M) to deionized water.

  In each experiment, a 5 ml aliquot of one of the above solutions was heated to 37 ° C. A portion of the appropriate salt (LMT.2MsOH, LMT.2HCl or LMT.2HBr) was added and the mixture was stirred for a while to completely dissolve the solid. This step was repeated until no further dissolution occurred.

  The results are shown in the following table:

  As you can see, LMT. 2MsOH has good water solubility.

ii) LMT. PH Dependence of 2MsOH Salt In related experiments, the following three buffer stock solutions were prepared (pH 2, pH 3, and pH 7):

pH 2 buffered aqueous solution A solution of potassium chloride (KCl) (0.2 M) (0.745 g in 50 mL of deionized water) was first prepared. 50 mL was taken from this solution and diluted with about 80 mL deionized water. A (0.2 M) hydrochloric acid (HCl) solution was then used to adjust the pH to 2, followed by further dilution with deionized water to a total of 200 mL. The final pH of 2.00 at 21.6 ° C. was recorded.

pH 3 Buffered Aqueous Solution A solution of potassium hydrogen phthalate (0.1 M) (2.042 g in 100 mL of deionized water) was first prepared. 100 mL was taken from this solution and diluted with about 50 mL of deionized water. A 0.2 M HCl solution was then used to adjust the pH to 3, followed by further dilution with deionized water to a total of 200 mL. The final pH of 2.99 at 21.7 ° C. was recorded.

pH 7 buffered aqueous solution A solution (0.1 M) of primary potassium phosphate (KH ₂ PO ₄ ) (1.370 g in 100 mL of deionized water) was first prepared. 100 mL was taken from this solution and diluted with about 80 mL of deionized water. A 0.5 M sodium hydroxide (NaOH) solution was then used to adjust the pH to 7, followed by further dilution with deionized water to a total of 200 mL. The final pH of 7.07 at 22 ° C was recorded.

Method A 5 mL aliquot of aqueous buffer solution was added to a vial containing micro-flea. The vial was placed in a water bath set at 25 ° C. LMT. 2M sOH was added. A 10 minute stirring time was allowed after each addition to ensure maximum chance for dissolution. The uniformity of the mixture was judged visually. After the stirring time, it was determined that the saturation point was reached when solids still existed as determined by visual inspection.

Results The viscosity of the resulting mixture made it impossible to properly isolate excess solids, so it was impossible to determine an accurate solubility. In conclusion, each result shows that the LMT. 2MsOH constitutes the lower limit, and LMT. The total mass of 2MsOH is reported as the range providing the upper limit.

  Results from three experiments are shown below:

  As can be seen, the solubility decreases slightly as pH decreases, but LMT. 2MsOH performed well in each of the three aqueous systems.

  In conclusion, LMT. 2MsOH exhibits better water solubility than MTC (not shown) and has enhanced solubility compared to the corresponding chloride and bromide salts. This suggests even greater utility for the treatments and uses disclosed herein.

Example 3 Method of Inhibiting Aggregation and Toxicity : Solid Phase Assay for Tau Aggregation The tau-tau aggregation assay uses purified recombinant tau fragments in a solid phase immunoassay. The method is described in detail, for example, in PCT International Patent Application Publication No. WO 96/30766. That is, this assay measures the binding of cleaved tau (amino acids 297-391) in solution to cleaved tau (residues 297-390) bound to the solid phase. The former binding is detected by antibody mAb 423 that specifically recognizes a peptide containing the C-terminal Glu-391 residue. In vitro formed tau complexes are pathological tau-tau binding interactions through 94 / 95-amino acid repeat domains (residues 297-390) found in the proteolytic stable core of paired helical fibrils It resembles the aggregated complex that forms in Alzheimer's disease as a result of the stability of action.

The B ₅₀ value (expressed as mean ± SE) is determined as the concentration of compound at which the tau-tau binding is reduced by 50%.

Methods: Cell-based tau aggregation assay This assay shows full-length human tau protein (htau40) under the control of an inducible promoter (pOPRSVI) and low levels of truncation under the control of a constitutive promoter (pcDNA3.1) Based on 3T6 mouse cells genetically engineered to express tau (295-390, dGA). Large amounts of htau40 expression is induced by the addition of IPTG (10-50 μM), which is then further cleaved by the process of full-length tau aggregation and processing in the presence of dGA tau acting as a template. Bring about generation. The addition of tau-tau aggregation inhibitors to the assay blocks this process. The method is described in more detail in PCT International Patent Application Publication No. WO 02/055720.

Results are expressed as the concentration at which the production of 12 kD fragment is inhibited by 50%. This is called the EC ₅₀ value.

Cells (4A and its clones) were grown to approximately 80% confluence in 10 cm dishes, then split into two 24-well plates and grown for 24 hours. Specimens are added at various concentrations, and after 24 hours, IPTG is added. After overnight incubation, the media was removed, the wells were washed with PBS, and the cells were collected by the addition of Laemmli buffer. Samples were stored at −20 ° C. for subsequent gel electrophoresis, western blotting and antibody labeling. Samples are separated by SDS-PAGE, transferred to a PVDF membrane, and tau labeled with the 7/51 antibody is detected by ECL at Kodak Image Station. Compounds are typically tested in triplicate at four concentrations over a series of concentration ranges and all samples are run on one gel. The intensity ratio of dGA to htau40 band, normalized to the control sample without drug, was plotted against drug concentration, and EC ₅₀ values were determined graphically from the concentration at which the ratio was 0.5.

The above method is summarized in Table 1 immediately below. In all experiments, MTC (TRx0014.047) was run as a control and EC ₅₀ values were normalized to MTC with EC ₅₀ = 0.59 μM.

Method: Cytotoxicity assay Cells (3T6 mouse fibroblasts) were grown to approximately 80% confluence in 10 cm dishes and then split into 96-well plates with 10% of 10 cm dishes per 96-well plate, 50 μl per well did. One row of 8 wells was left empty (to be a reagent blank in the assay). After the cells are grown overnight, they are added to 4 wells at the starting concentration (typically 200 μM for MTC or LMT.2HBr), and in subsequent wells, serially diluted 1: 2 and the last 4 of the cells Wells were used as drug-free controls. This makes it possible to test two drugs per 96 well plate. Cells were left in the presence of drug for 48 hours, after which the medium was removed and the cells were washed with PBS. Cell numbers were determined using the Cytotox 96 well kit (Promega) based on the lactate dehydrogenase (LDH) assay. The assay quantitatively measures LDH, a stable cytoplasmic enzyme released by cell lysis. The released LDH is measured by an enzymatic assay that results in conversion of the tetrazolium salt to the red formazan product. The amount of color produced is proportional to the number of lysed cells. That is, cells are lysed for 45-60 minutes with 50 μl / well of 1 × lysis buffer, followed by 30 minutes of 50 μl / well LDH assay reagent and 50 μl / well stop buffer. Absorbance is read at 490 nm. The relative absorbance for untreated wells (untreated cells = 1.0) was plotted against drug concentration. From the graph, the LD ₅₀ was determined from the concentration at which the absorbance decreased by 50%. LMT. During the 2HBr test, MTC (TRx0014.047) was run as a control in all experiments and LD ₅₀ values were calibrated against MTC with LD ₅₀ = 65 μM.

result:
Various bis (sulfonate) salts according to the present invention were tested and bis (halide) salt N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diaminium dichloride (LMTC, LMT.2HCl) And N, N, N ′, N′-tetramethyl-10H-phenothiazine-3,7-diaminium di (bromide) LMT. 2HBr and methylthioninium chloride (MTC) were compared.

  In vitro data for different methylthioninium salt forms are summarized in Table 2 immediately below:

Comment LMT. 2MsOH and LMT. The EC50 values (mean ± SE) for 2HCl are 0.19 ± 0.04 μM and 0.63 ± 0.10 μM, respectively, and the corresponding therapeutic indices are 179 and 102.

  The relative potency of compounds in a cell-based model of tau-tau aggregation is reported in LMT. 2MsOH> MTC, LMT. 2HBr, LMT. 2HCl. Compared to MTC, LMT. The therapeutic index of 2MsOH is 63% higher.

  The order of potency in the cell-based assay is MTC, LMT. 2MsOH> LMT. 2HCl> LMT. 2HBr. LMT. 2MsOH and LMT. The B50 values for 2HCl are 238.2 ± 74.2 μM and 360.8 ± 38.2 μM, respectively. The order of relative potency in the cell-free assay is LMT. 2MsOH> MTC, LMT. 2HCl, LMT. 2HBr.

Example 4-Toxicology, Impurities and Effects on Hematopoietic System LMT. 2HBr, LMT. 2HCl, LMT. 2MsOH or MTC was administered daily to female Wistar rats for 14 days; doses were 95 mg MT / kg / day from day 1 to day 10 and 60 mg MT / kg / day from day 11 to day 14. there were. Clinical signs of raised body posture, behavioral depression, and general weakness were seen in all treatment groups. Treatment-related death is caused by LMT. Occurs in 2HBr and MTC treatment groups.

  Changes in red blood cell parameters were seen in the blood and bone marrow of all treatment groups, which was an indicator of regenerative anemia. These included the following: a decrease in the number of red blood cells in the blood, an increase in low concentrations of hemoglobin and reticulocytes, and an increase in the number of erythroid progenitors in the bone marrow. This was demonstrated pathologically by increasing the level of erythropoiesis in the spleen.

  Although a decrease in the number of neutrophilic granulocytes was seen in the bone marrow of all treated animals, the strength of this effect was greater than that of the other groups. It was quite large in the 2HBr treatment group. This difference is also observed in the severity of neutropenia observed in the prepared blood smears. There is a marked depletion of mature neutrophils in 2HBr treated animals, a moderate decrease in MTC, and LMT. 2HCl or LMT. There was no decrease in the 2MsOH group. The results of this study show that at least in rats, LMT. 2HBr, LMT. 2HCl, LMT. It suggests a higher tendency to cause neutrophil depletion than 2MsOH or MTC. A decrease in the number of mature neutrophils and granulocytes is also observed in LMT. It was observed in the bone marrow at a high dose (45 mg MT / kg / day) in a 6 month study of 2HBr. LMT. The neutropenia or neutropenia observed after 2HBr, although reversible, will make patients more susceptible to bacterial infections because its primary role is bacterial destruction.

  Thus, for tolerability (dose-dependent death) and neutrophil response in rats, LMT. 2MsOH is LMT. Shows improved properties compared to 2HBr.

  LMT. 2HCl and LMT. 2MsOH was comparable in the above analysis, but there were differences in the impurities found in the two salt forms. For LMT, 2HCl, the presence of methyl chloride was detected during the synthesis and trapped in the product in a manner that was difficult to remove completely. In contrast, LMT. During the synthesis of 2MsOH, impurities such as ethyl and methyl methanesulfonate (EMS, MMS) could be controlled to much lower levels.

  Studies on the hematopoietic system were performed in rats, monkeys and minipigs.

  The lowest doses at which methemoglobinemia was observed were 15 mgMT / kg / day (MTC and LMT.2HBr) or 30 mgMT / kg / day (LMT.2MsOH) in rats, 5.3 mgMT / kg / day (MTC) in primates ), And 10 mg MT / kg / day (LMT.2MsOH and LMT.2HBr) in minipigs.

  Nine months of LMT. In the 2MsOH study, after the first 28 days of dosing, there was no sign of methemoglobinemia at 3 mg MT / kg / day.

  However, as expected, MTC, LMT. 2HBr or LMT. As the 2MsOH dose level increases, RBCs are evidenced by an increase in methemoglobin levels and the formation of Heinz bodies (aggregates of denatured and precipitated hemoglobin in erythrocytes) at doses that did not ultimately tolerate Signs of oxidative stress on the skin appeared in a dose-dependent manner.

Example 5-Pharmacokinetics Figure 18 shows two oral doses (2 and 15 mg / kg) of LMT. 2HBr, LMT. 2HCl and LMT. 2 shows a comparison of the time course of plasma concentration in the MT portion after 2MsOH administration.

As you can see, LMT. The C _max (at 1 hour T _max ) for 2MsOH is LMT. 2HCl or LMT. Two times higher than that for 2HBr. Therefore, LMT. 2MsOH is LMT. 2HCl or LMT. It can provide more effective exposure to MT than 2HBr.

Example 6-Gastric irritation test study (28-day-old rats according to MTC or LMT.2HBr): Occurrence and severity of selected gastric microscopic findings from the final animal

  From the above, the following can be predicted with 10 animals per group.

  Test (28-day-old rats with LMT.2MsOH): Development and severity of microscopic findings of selected sternum, liver, spleen and stomach from the final animal

  These results are shown in LMT. 2MsOH was added to LMT. It indicates that stomach irritation is less likely to occur than 2HBr.

Example 7-Formulation
Formulation Example 1: Preparation of LMTM tablet using direct tableting method Tablets having the following composition were prepared by direct tableting method.

  LMTM, spray dried mannitol, microcrystalline cellulose, crospovidone and magnesium stearate were mixed in a tumbling blender and then compressed using a tablet press.

The tablet cores were then film coated with an aqueous suspension of Opadry ^* Blue ( ^* registered for the Colorcon series of film coating materials).

Formulation Example 2-Preparation of LMTM tablet by dry granulation method (roller compression) Tablets having the following composition were prepared by dry granulation method:

  LMTM, spray dried mannitol, microcrystalline cellulose, crospovidone and magnesium stearate were mixed in a tumbling blender. The mixture was then granulated using a roller compressor and ground with a vibratory granulator using a suitable screen. In this case, half of the magnesium stearate was used before roller compaction, and then half of the magnesium stearate was added to the granulation process and mixed prior to compression in a conventional tablet press.

The tablet cores were then film coated with an aqueous suspension of Opadry ^* Blue ( ^* registered for the Colorcon series of film coating materials).

Formulation Example 3-Preparation of LMTM tablets by dry granulation (slagging) Tablets having the following composition were prepared by further dry granulation methods:

  LMTM and excipients were mixed in a tumbling blender and then compressed using a tablet press to produce slugs (plain, flat surface tablets).

  The slug was then pulverized using a vibratory granulator equipped with a 20 mesh screen.

  In this example, half of the magnesium stearate was used before slagging, and then half of the magnesium stearate was added to the granulation process and mixed prior to compression in a conventional tablet press.

The tablet cores were then film coated with an aqueous suspension of Opadry ^* Blue ( ^* registered for the Colorcon series of film coating materials).

Formulation Example 4: Preparation of LMTM tablets by wet granulation of excipients and further incorporation of LMTM while granulating Tablets having the following composition were prepared by a wet granulation method:

  Mannitol, crospovidone (1/3 of the total) and microcrystalline cellulose were mixed in a tumbling blender. The mixed material was then granulated using a PVP solution in water. The wet mass was dried in a fluid bed dryer and ground using a vibratory granulator equipped with a suitable screen.

The ground material was then mixed with the remaining crospovidone and magnesium stearate and LMTM prior to compression in a conventional tablet press. The tablet cores were then film coated with an aqueous suspension of Opadry ^* Blue ( ^* registered for the Colorcon series of film coating materials).

Formulation Example 5: Preparation of LMTM capsule A capsule having the following composition was prepared.

  LMTM and excipients were mixed in a tumbling blender. The resulting drug mixture was filled into capsules (50, 75, 100, 125 and 150 mg formulations into size 1 capsules and 200 mg formulation into size 0 capsules) using a capsule filling machine. Both gelatin capsules and HPMC capsules were prepared.

Formulation Example 6: Results of stability test of LMTM 75 mg film-coated tablet

Formulation Example 7: Results of stability test of LMTM 100 mg film-coated tablet

Formulation Example 8: Results of stability test of LMTM 75 mg film-coated tablet

Formulation Example 9: Results of stability test of LMTM 100 mg film-coated tablet

Formulation Example 10: LMTB 100 mg film-coated tablet

Formulation Example 11: LMTM 75 mg film-coated tablet

Formulation Example 12-Dissolution Test LMTB film-coated tablets (3 x 100 mg) and LMTM tablets (4 x 75 mg) prepared as in Formulation Examples 10 and 11 were agitated at a paddle speed of 50 rpm (see Figure 19) ), Standard pharmacopoeia methods (USP34) and the conditions specified below.

  The results are shown in the table below.

Tablets stored for various periods under normal conditions (25 ° C./60% RH) or “stress” conditions (40 ° C./75% RH) were also tested using the same method.
The results are shown in the table below.

Appendix-Crystallographic data
LMT. Crystallographic data for EDSA (Figure 17a)

LMT. Crystallographic data for 2EsOH (Figure 17b)

LMT. Crystallographic data for 2MsOH (Figure 17c)

Claims (71)

The following general formula (I):
{Where
R ¹ and R ⁹ are each independently selected from —H, C _1-4 alkyl, C _2-4 alkenyl and halogenated C _1-4 alkyl;
R ^3NA and R ^3NB are each independently selected from —H, C _1-4 alkyl, C _2-4 alkenyl and halogenated C _1-4 alkyl;
R ^7NA and R ^7NB are each independently selected from —H, C _1-4 alkyl, C _2-4 alkenyl and halogenated C _1-4 alkyl;
Furthermore, where
R ^A and R ^B are each independently selected from C _1-4 alkyl, halogenated C _1-4 alkyl, and C _6-10 aryl; or R ^A and R ^B are connected to form C Form a group selected from _1-6 alkylene and C _6-10 arylene}
And a pharmaceutically acceptable salt thereof. The compound according to claim 1, wherein R ^A and R ^B are each independently C _1-4 alkyl. The compound of claim 1, wherein R ^A and R ^B are each independently methyl. The compound according to any one of claims 1 to 3, wherein R ¹ and R ⁹ are each independently -H. The compound according to any one of claims 1 to 4, wherein R ^3NA and R ^3NB are each independently C _1-4 alkyl. ^6. The compound of claim 5, wherein ^R3NA and ^R3NB are each independently methyl. The compound according to any one of claims 1 to 6, wherein R ^7NA and R ^7NB are each independently C _1-4 alkyl. ^8. The compound of claim 7, wherein ^R7NA and ^R7NB are each independently methyl. The following formula:
Or a pharmaceutically acceptable salt, solvate or hydrate thereof.   10. A compound according to any one of claims 1 to 9, which is in crystalline form.   10. A compound according to claim 9 in crystalline form having the crystal structure represented by FIGS.   12. A compound according to any one of claims 1 to 11, which is in a substantially purified form.   A composition comprising a compound according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier or diluent.   A method for preparing a pharmaceutical composition comprising the step of mixing a compound according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier or diluent.   14. A pharmaceutical composition according to claim 13 in solid dosage form comprising a compound according to any one of claims 1 to 12 and at least one diluent suitable for dry compression.   The composition of claim 15, wherein the solid dosage form is produced by direct tableting.   The solid dosage form is produced by dry granulation. The composition according to claim 15.   18. The solid dosage form of any of claims 15-17, wherein the solid dosage form elutes to at least 80% within 30 minutes as assessed by standard pharmacopoeia methods as defined in the United States Pharmacopeia (USP) General Rules <711>. The composition according to claim 1.   The at least one diluent is selected from microcrystalline cellulose, lactose, mannitol, calcium salts such as dicalcium phosphate, calcium sulfate, calcium carbonate, and sugars such as lactose, sucrose, dextrose and maltodextrin. The composition according to any one of 15 to 18.   20. A composition according to any one of claims 15 to 19, further comprising at least one disintegrant.   21. The composition of claim 20, wherein the disintegrant is selected from cross-linked polyvinyl pyrrolidone (crospovidone), sodium starch glycolate, cross-linked sodium carboxymethylcellulose (croscarmellose sodium), or pregelatinized starch.   The composition according to any one of claims 15 to 21, wherein the solid dosage form is film coated.   23. The composition of any one of claims 15-22, further comprising an acid having a pK1 greater than 1.5.   24. The composition of claim 23, wherein the acid is selected from maleic acid, phosphoric acid, ascorbic acid, sorbic acid, aspartic acid and sialic acid.   25. The composition of claim 23 or claim 24, further comprising a carrier selected from mannitol, cellulosic material, starch or mixtures thereof.   26. A composition according to any one of claims 15 to 25, characterized in that the composition comprises particles, and more than 10% of the particles have a size greater than 10 microns.   26. A composition according to any one of claims 15 to 25, comprising a carrier having a water-avoiding particle shape such as Elcema ™, ethylcellulose, mannitol, or Starch 1500 ™.   Free-flowing cohesiveness comprising a compound according to any one of claims 1 to 12 and at least one diluent suitable for dry compression and optionally one or more other excipients. The powder, wherein the powder is compressible into a solid dosage form.   13. Dry compression of a dense powder mixture comprising a compound according to any one of claims 1-12 and at least one diluent suitable for dry compression and optionally one or more other excipients. 15. A method according to claim 14 for the manufacture of a pharmaceutical composition.   30. The method of claim 29, wherein the method comprises a direct tableting method, wherein the compound, at least one diluent, and optional excipients are mixed together in a solid particulate form. Producing the intimate mixture and then compressing using a tablet press.   The at least one diluent is selected from microcrystalline cellulose, lactose, mannitol, calcium salts such as dicalcium phosphate, calcium sulfate and calcium carbonate, and sugars such as lactose, sucrose, dextrose and maltodextrin. 30. The method according to 30.   32. The method of claim 30, wherein the at least one diluent is a granulated product prepared by wet granulation of one or more excipients.   35. The method of claim 32 comprising the steps of wet agglomeration of one or more excipients using a granulation liquid and drying to form the granulation product.   The method includes a dry granulation method, wherein the compound, at least one diluent, and optional excipients are formed into a compressed mass, crushed and then compressed using a tablet press. 30. The method of claim 29.   34. A method according to any one of claims 28 to 33, further comprising applying a film coating to the resulting tablet.   28. A compound or composition according to any one of claims 1 to 12 or 15 to 27 for use in a method of treatment or prevention of the human or animal body by therapy.   28. A compound or composition according to any one of claims 1 to 12 or 15 to 27 for use in a method for the treatment or prevention of protein aggregation disorders.   28. A compound or composition according to any one of claims 1 to 12 or 15 to 27 for use in a method of treating or preventing tauopathy.   Alzheimer's disease (AD), Pick's disease, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), FTD with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobe Degeneration (FTLD) syndrome, disinhibition / dementia / parkinsonism / muscular atrophy complex (DDPAC), pallidum bridge substantia nigra degeneration (PPND), Guam ALS syndrome, palluitous substantia nigra degeneration (PNLD) ), Cerebral cortex basal ganglia degeneration (CBD), taste-granular dementia (AgD), boxer dementia (DP) or chronic traumatic brain injury (CTE), Down syndrome (DS), Lewy body dementia ( DLB), subacute sclerosing panencephalitis (SSPE), MCI, C type Niemann-Pick disease (NPC), type B Sanfilip syndrome (MPS III B), or myotonic dystrophy (DM 28) A compound or composition according to any one of claims 1-12 or 15-27 for use in a method of treatment or prevention of DM1 or DM2.   28. A compound or composition according to any one of claims 1 to 12 or 15 to 27 for use in a method of treating or preventing Alzheimer's disease.   28. A compound or composition according to any one of claims 1 to 12 or 15 to 27 for use in a method of treating or preventing Parkinson's disease.   28. A compound or composition according to any one of claims 1 to 12 or 15 to 27 for use in a method of treating or preventing PSP, ALS, or FTLD.   Claims for use in methods of treatment or prevention of other polyglutamine diseases such as Huntington's disease or bulbar spinal muscular atrophy (Kennedy's disease), dentate nucleus red nucleus pallidus atrophy or spinocerebellar ataxia Item 13. The compound according to any one of Items 1 to 12.   28. A compound or composition according to any one of claims 1-12 or 15-27 for use in a method of treating or preventing skin cancer or melanoma; or viral, bacterial or protozoan disease states.   45. A compound or composition for use according to claim 44, wherein the viral, bacterial or protozoal disease is selected from hepatitis C, HIV, West Nile virus (WNV) and malaria.   28. Use of a compound or composition according to any one of claims 1 to 12 or 15 to 27 in the manufacture of a medicament for use in the treatment or prevention of protein aggregation disorders.   28. Use of a compound or composition according to any one of claims 1-12 or 15-27 in the manufacture of a medicament for use in the treatment or prevention of tauopathy.   Alzheimer's disease (AD), Pick's disease, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), FTD with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobe Degeneration (FTLD) syndrome, disinhibition / dementia / parkinsonism / muscular atrophy complex (DDPAC), pallidum bridge substantia nigra degeneration (PPND), Guam ALS syndrome, palluitous substantia nigra degeneration (PNLD) ), Cerebral cortex basal ganglia degeneration (CBD), taste-granular dementia (AgD), boxer dementia (DP) or chronic traumatic brain injury (CTE), Down syndrome (DS), Lewy body dementia ( DLB), subacute sclerosing panencephalitis (SSPE), MCI, type C Niemann-Pick disease (NPC), type B Sanfilip syndrome (or mucopolysaccharidosis IIIB), or myotonic dystrophy (DM), in the manufacture of a medicament for use in the treatment or prophylaxis of DM1 or DM2, the use of a compound or composition according to any one of claims 1 to 12 or 15 to 27.   28. Use of a compound or composition according to any one of claims 1-12 or 15-27 in the manufacture of a medicament for use in the treatment or prevention of Alzheimer's disease.   28. Use of a compound or composition according to any one of claims 1-12 or 15-27 in the manufacture of a medicament for use in the treatment or prevention of Parkinson's disease.   28. Use of a compound or composition according to any one of claims 1-12 or 15-27 in the manufacture of a medicament for use in the treatment or prevention of PSP, ALS or FTLD.   Manufacture of pharmaceuticals for use in the treatment or prevention of other polyglutamine diseases such as Huntington's disease or bulbar spinal muscular atrophy (Kennedy's disease), dentate nucleus red nucleus pallidal atrophy and spinocerebellar ataxia Use of a compound or composition according to any one of claims 1 to 12 or 15 to 27.   28. A compound according to any one of claims 1-12 or 15-27 in the manufacture of a medicament for use in the treatment or prevention of skin cancer or melanoma; or viral, bacterial or protozoal disease states Use of the composition.   54. Use according to claim 53, wherein the viral, bacterial or protozoal disease is selected from hepatitis C, HIV, West Nile virus (WNV) and malaria.   28. Treatment or prevention of a protein aggregation disease in a subject comprising administering to the subject a prophylactically or therapeutically effective amount of a compound or composition according to any one of claims 1-12 or 15-27. Method.   28. A method for treating or preventing tauopathy in a subject comprising administering to the subject a prophylactically or therapeutically effective amount of a compound or composition according to any one of claims 1-12 or 15-27.   28. A method of treating or preventing Alzheimer's disease in a subject comprising administering to the subject a prophylactically or therapeutically effective amount of a compound or composition according to any one of claims 1-12 or 15-27.   28. A method of treating or preventing Parkinson's disease in a subject comprising administering to the subject a prophylactically or therapeutically effective amount of a compound or composition according to any one of claims 1-12 or 15-27.   Treatment of PSP, ALS or FTLD in said subject comprising administering to the subject a prophylactically or therapeutically effective amount of a compound or composition according to any one of claims 1-12 or 15-27, or Prevention method.   28. Huntington's disease or bulbar spinal muscular atrophy in said subject comprising administering to the subject a prophylactically or therapeutically effective amount of a compound or composition according to any one of claims 1-12 or 15-27. For the treatment or prevention of other polyglutamine diseases such as erythematosus (Kennedy's disease), erythrocytic red nuclei Ryukyu atrophy or spinocerebellar ataxia.   A skin cancer or melanoma in said subject comprising administering to the subject a prophylactically or therapeutically effective amount of a compound or composition according to any one of claims 1-12 or 15-27; or a virus For the treatment or prevention of sexual, bacterial or protozoan diseases.   62. The method of claim 61, wherein the viral, bacterial or protozoal disease is selected from hepatitis C, HIV, West Nile virus (WNV) and malaria.   28. A method of reversing and / or inhibiting protein aggregation comprising contacting the protein with an effective amount of a compound or composition according to any one of claims 1-12 or 15-27. , Said method.   A method of reversing and / or inhibiting protein aggregation in a mammalian brain, wherein said aggregation is associated with a disease state and said treatment is directed to said mammal in need of said treatment. 28. The method comprising administering a prophylactically or therapeutically effective amount of a compound or composition according to any one of 1-12 or 15-27. A method for inactivating pathogens in a sample comprising:
13. The method comprising: introducing the compound of any one of claims 1-12 into the sample; and then exposing the sample to light. 13. A process for the preparation of a compound according to any one of claims 1 to 12 from N-protected 3,7-diamino-10H-phenothiazine, comprising:
Ring amino deprotection (DP) step; and salt formation (SF) step. Said N-protected 3,7-diamino-10H-phenothiazine has the following formula (II):
{Where
R ^Prot is an amine protecting group;
R ¹ , R ⁹ , R ^3NA , R ^3NB , R ^7NA , R ^7NB , R ^A and R ^B are as defined above}
68. The method of claim 66, wherein the compound is 68. The method of claim 67, wherein ^RProt is acyl.   69. A method according to any one of claims 66 to 68, wherein ring amino deprotection (DP) and salt formation (SF) are performed simultaneously.   70. The method of claim 69, wherein ring amino deprotection (DP) and salt formation (SF) comprises treatment of the N-protected 3,7-diamino-10H-phenothiazine with at least one sulfonic acid.   Ring amino deprotection (DP) and salt formation (SF) methanesulfone of said N-protected 3,7-diamino-10H-phenothiazine in organic solvent to produce bis (methanesulfonate) salt 71. The method of claim 70, comprising treatment with acid and water.